Cholinergic enhancers with improved blood-brain barrier permeability for the treatment of diseases accompanied by cognitive impairment

ABSTRACT

A method for the treatment of a neurodegenerative, psychiatric or neurological disease associated with a cholinergic deficit comprising administering GLN-1062 or a pharmaceutically acceptable salt thereof by nasal administration to a patient in need thereof.

BACKGROUND OF THE INVENTION Field of the Invention

The disclosure refers to compounds that, in addition to enhancing thesensitivity to acetylcholine and choline, and to their agonists, ofneuronal cholinergic receptors, and/or acting as cholinesteraseinhibitors and/or neuroprotective agents, have enhanced blood-brainbarrier permeability in comparison to their parent compounds. Thecompounds are derived (either formally by their chemical structure ordirectly by chemical synthesis) from natural compounds belonging to theclass of amaryllidaceae alkaloids e.g., Galantamine, Narwedine andLycoramine, or from metabolites of said compounds. The compounds of thepresent invention can either interact as such with their targetmolecules, or they can act as “pro-drugs”, in the sense that afterreaching their target regions in the body, they are converted byhydrolysis or enzymatic attack to the original parent compound and reactas such with their target molecules, or both. The compounds of thisdisclosure may be used as medicaments for the treatment of human braindiseases associated with a cholinergic deficit, including theneurodegenerative diseases Alzheimer's and Parkinson's disease and theneurological/psychiatric diseases vascular dementia, schizophrenia andepilepsy. Galantamine derivatives disclosed herein have higher efficacyand lower levels of adverse side effects in comparison to galantamine,in treatment of human brain diseases.

Description of the Related Art

The diffusion of compounds from the blood plasma into the brain iscomplicated by the presence of the blood-brain barrier that is amembrane that segregates the brain interstitial fluid from thecirculating blood. In designing drugs active in the central nervoussystem and able to cross the blood-brain barrier, one can exploitendogenous active mechanisms, utilize proper delivery techniques ormodify the chemical structure through the synthesis of pro-drugderivatives.

Galantamine is an alkaloid that can be isolated from the bulbs ofvarious snowdrop (Galanthus) and narcissus species (daffodils,Amaryllidaceae), and recently in particularly high concentrations fromLycoris radiata, and related species. Synthetic Galantamine hydrobromideis manufactured by, among other companies, Sanochemia and JanssenPharmaceutica. The drug has been approved in more than 70 nations forthe treatment of mild-to-moderate Alzheimer's disease (AD), aneurodegenerative brain disease. Extensive studies of thepharmacokinetic profile, tissue distribution and accumulation ofGalantamine in mice, rats, rabbits and dogs have shown that Galantaminegiven orally is by no means preferentially distributed to the brainwhere it is supposed to exert its therapeutic activity in said braindiseases. In contrast, it is accumulated at much higher concentrationsin other body tissues. In male and female rat tissues the highestconcentrations are observed in kidney (tissue to plasma ratio; T/P˜10-15), salivary and adrenal gland (T/P ˜7-14), female rat spleen (T/P˜20), lung, liver, heart, skeletal muscle and testes (T/P ˜2-4). Incontrast, the brain to plasma ratio is only T/P ˜1.5. Similarly, thebrain/plasma partition coefficient Kbrain is significantly lower thanmost other Korgan of Galantamine.

Limited penetration ability of Galantamine through the blood-brainbarrier (BBB) into the central nervous system (CNS) is indicated also bythe compound's log P value of 1.3, log P being defined as the decadiclogarithm of the partition coefficient P which is the ratio of theconcentration of compound in aqueous phase to the concentration ofcompound in immiscible solvent, as the neutral molecule. The log P valueis obtained by predictive computational methods and provides a generalguideline as to whether a drug gains rapid access to the CNS, or not.Thus, it has been established over the past more than 30 years that,assuming passive absorption, drugs with optimum CNS penetrationgenerally have log P values around or somewhat above 2. Significantlylower log P values are often associated with low brain-to-plasma andhigh non-brain tissue-to-plasma ratios (see above: log P and T/P ratiosfor Galantamine). However, much higher log P values are also ofdisadvantage, as high lipophilicity is often associated with toxicity,non-specific binding, insufficient oral absorption and limitedbioavailability. It follows from this account that BBB penetration andT/P ratios are essential parameters to be considered in the case ofdrugs that are supposed to act mainly or exclusively in the centralnervous system.

Other important parameters controlling BBB penetration of a compound arethe total polar surface area, the existence of ionizable groups on themolecule and the affinity of binding to biological membranes as comparedto the affinity of binding to serum albumin. The latter data set isoften used to scrutinize calculated log P values. In those cases inwhich special transport systems do not play a major role for thetransport of a compound through the BBB, the predictions oflipophilicity and BBB penetration properties are quite suitable for thedesign of derivatives that transfer the BBB more efficiently than theparent compound.

The present disclosure relates to methods by which the lipophilicityand/or BBB penetration and/or brain-to-plasma ratio of a compound isenhanced by formation of a reversible linkage with one or more suitablegroups so as to yield “pro-drugs”, i.e., chemical derivatives that,after having passed through the blood-brain barrier, are converted(back) to the original compound itself inside the patients brain.Liberation of the parent compound may be by chemical hydrolysis orenzymatic attack, or by redox reactions. In another embodiment, thepresent invention refers to compounds that after chemical modificationof the base compound have achieved a log P value more favourable for BBBpenetration, with these derivatives acting as such at their targetmolecules in the patient's brain.

The plant alkaloid galantamine has been described as a cholinesteraseinhibitor (ChE-I) and as a nicotinic acetylcholine receptor (nAChR)sensitizing agent (APL; allosterically potentiating ligand), andgalantamine has been proposed for the treatment of several human braindiseases, including Alzheimer's disease (AD). Presently, the complianceof Alzheimer patients to treatment with ChE-I and APL is rather low, ofthe order of 20%, a key reason being the adverse effects nausea,diarrhea, vomiting, anorexia and muscle cramps. In the case ofgalantamine, the majority of these adverse effects is due to actions ofthe drug while passing through the gastro-intestinal tract, and to itsrather limited permeation through the blood-brain barrier (BBB) into thebrain. To help patients coping with the adverse effects of galantamine,the manufacturer's recommended daily dose of the drug is limited to16-24 mg per day, and this dose is slowly reached by stepwise doseincrease, beginning at 4 mg/day and over a period of 2-3 months.

The rather low levels of accumulation of galantamine in the brain, whenadministered as the unmodified drug, are a serious disadvantage withrespect to the drug's therapeutic use, i.e., for the treatment ofcognitive disorders, such as AD. As indicated by the brain-to-plasmaratio of ˜1.3, only a small part of the administered drug reaches thebrain, and the high levels of the drug in other (peripheral) tissuescause most, if not all, of the observed adverse effects. The mostlyperipheral action of galantamine is also indicated in its previous usefor the treatment of a number of neuromuscular disorders, includingMyasthenia gravis and poliomyelitis.

In WO2007/039138 reference is made to the low hydrophobicity and relatedlimited partition into the human brain of galantamine, and severalprocedures for overcoming these drawbacks of a medication that issupposed to act on target molecules located in the brain's centralnervous system are proposed. In the same document numerous derivativesof galantamine that significantly improve transport of the respectivecompound through the blood-brain barrier (BBB) are described and theyare proposed as drugs for the treatment of a variety of diseasesassociated with cognitive deficits.

Presently approved drugs for the treatment of Alzheimer's disease (AD)have in common that they all target excitatory neurotransmission in thebrain, namely the cholinergic and the glutamatergic systems. Three ofthe four presently available drugs (Donepezil, Rivastigmin, Galantamine,Memantine) are cholinergic enhancers (Donepezil, Rivastigmin,Galantamine) in that they all inhibit the family ofacetylcholine-degrading enzymes denoted as cholinesterases (ChE).Inhibition of ChE increases the synaptic concentrations of acetylcholine(ACh), thereby enhancing and prolonging the action of ACh on muscarinic(mAChR) and nicotinic (nAChR) acetylcholine receptors. In addition toacting as ChE inhibitor, Galantamine also acts by allostericallystimulating (sensitizing) cholinergic receptors. Allostericsensitization of nicotinic receptors enhances their activation by ACh orcholine (Ch), thereby correcting for a disease-associated deficit intransmitter or receptor concentration (Maelicke A & Albuquerque E X(1996) Drug Discovery Today 1, 53-59; Maelicke A & Albuquerque E X(2000) Eur J Pharmacol 393, 165-170). In addition to their therapeuticbenefits, these drugs induce adverse peripheral and central sideeffects; the muscarinic ones including nausea, vomiting and diarrhea,and the nicotinic ones including tremors and muscle cramps. From metadata (Cochrane reviews, (2004), Issue 4) and direct comparison clinicalstudies (Wilcock G K et al. (2000) Brit Med Journ 321:1-7), therelatively weakest of the three presently used ChE inhibitors,Galantamine, has the highest clinical efficacy, with the therapeuticbenefit achieved at concentrations that are well below those requiredfor effective inhibition of AChE (Raskind M A et al. (2000) Neurology54, 2261-2268; Maelicke A & Albuquerque E X (2000) Eur J Pharmacol 393,165-170). It has been suggested that the higher therapeutic efficacy ofGalantamine, as compared to the other two available ChE inhibitors, isdue to an additional or alternative mode of action, i.e., allostericsensitization of nAChR (Maelicke A & Albuquerque E X (1996) DrugDiscovery Today 1, 53-59).

Galantamine enhances nicotinic cholinergic neurotransmission by actingdirectly on nicotinic receptors (Schrattenholz A et al. (1996) MolPharmacol 49, 1-6; Samochocki M et al. (2003) J Pharmacol Exp Therap305, 1024-1036). The drug binds to a distinct allosteric site on thesereceptors (Schrider B et al. (1993) J Biol Chem 269, 10407-10416), fromwhich it acts synergistically with acetylcholine (or choline) tofacilitate nAChR activation (Maelicke A & Albuquerque E X (1996) DrugDiscovery Today 1, 53-59; Maelicke A & Albuquerque E X (2000) Eur JPharmacol 393, 165-170). Compounds acting like Galantamine in this wayare referred to as “allostericaly potentiating ligands (APL)”(Schrattenholz A et al. (1996) Mol Pharmacol 49, 1-6, Maelicke A &Albuquerque E X (2000) Eur J Pharmacol 393, 165-170).

The APL action on human nicotinic receptors has been demonstrated byelectrophysiological studies using human brain slices (Alkondon, M. etal., (2000) J Neurosci 20, 66-75) and human recombinant cell lines eachexpressing a single nAChR subtype (Samochocki M et al (2000) Acta NeuroScand Suppl 176, 68-73, Samochocki M et al. (2003) J Pharmacol ExpTherap 305, 1024-1036). All human nAChR subtypes analysed so far aresensitive to enhancement by APL. In the presence of Galantamine, thebinding affinity and channel opening probability of nAChR are increased,leading to a decrease in EC50 for ACh between 30% and 65% (Samochocki Met al (2000) Acta Neuro Scand Suppl 176, 68-73, Samochocki M et al.(2003) J Pharmacol Exp Therap 305, 1024-1036). Furthermore, Galantamineincreases the slope of the dose-response curve for ACh, which has beeninterpreted as an increase in the cooperativity between nAChR subunits(Maelicke A & Albuquerque E X (1996) Drug Discovery Today 1, 53-59).

The APL effect of Galantamine is observed at submicromolarconcentrations (Samochocki M et al (2000) Acta Neuro Scand Suppl 176,68-73, Samochocki M et al. (2003) J Pharmacol Exp Therap 305,1024-1036), i.e., below the concentration range at which ChE inhibitiontakes place. The two modes of action of nicotinic APL are independent ofeach other, as was shown by ion flux studies (Okonjo K et al (1991) EurJ Biochem 200, 671-677; Kuhlmann J et al (1991) FEBS Lett 279, 216-218)and electrophysiological studies of brain slices from both rats andhumans (Santos M D et al (2002) Mol Pharmacol 61, 1222-1234). In thesestudies, when cholinesterase activity was completely blocked by eitherreversible or irreversible blocking agents, the nicotinic APL, e.g.,Galantamine, still was able to produce an APL effect of the same size asin the absence of the other ChE inhibitors. Of the cholinesteraseinhibitors presently approved as AD drugs, Galantamine is the only onewith nicotinic APL activity (Maelicke A et al (2000) Behav Brain Res113, 199-206).

The use of Galantamine and other APL as a drug treatment strategy forcognitive disorders, including AD and PD was proposed in 1996 (MaelickeA & Albuquerque E X (1996) Drug Discovery Today 1, 53-59). Later, theproposal was extended to vascular and mixed dementia (Maelicke A et al(2001) Biol Psychiatry 49, 279-288), schizophrenia, epilepsy and otherdiseases with a nicotinic cholinergic deficit.

The comparatively low levels of accumulation of Galantamine in the brainare a serious disadvantage with respect to the drug's therapeutic use,i.e., for the treatment of cognitive disorders, such as AD. As indicatedby the T/P ratios, only a small part of the administered drug reachesthe brain, and the high levels of the drug in other (peripheral) tissuesmay be responsible for some of the observed adverse side effects. As apoint in case, long before having been approved for the treatment of AD,Galantamine has primarily been used for the treatment of a number ofneuromuscular disorders, including Myasthenia gravis and poliomyelitis.

EP-A 648 771, EP-A 649 846 and EP-A 653 427 all describe Galantaminederivatives, a process for their preparation and their use asmedicaments, however none of these applications considers ways and meansof enhancing penetration through the blood-brain barrier andbrain-to-plasma ratio of base compounds and derivatives.

U.S. Pat. No. 6,150,354 refers to several Galantamine analogues for thetreatment of Alzheimer's disease. However, selective chemicalmodification for the purpose of increasing penetration through theblood-brain barrier is not considered.

WO 01/74820, WO 00/32199 and WO 2005030333 refer to derivatives andanalogues of Galantamine for the treatment of a variety of human brainand other diseases, and acute functional brain damage. However,selective chemical modifications or other means of improving blood-brainbarrier penetration are not considered.

WO 88/08708, WO 99/21561, WO 01/43697 and US 2003/0162770 refer toderivatives and analogues of Galantamine for the treatment of variouscognitive symptoms. However, selective chemical modifications or othermeans of improving blood-brain barrier penetration are not considered.

WO 2005/030713 refers to a method for the synthesis of optical isomersof Galantamine from a Narwedine bromoamide derivative. However, it doesnot deal with other derivatives of Galantamine, or their use asmedicaments, or chemical modifications aimed at enhancing blood-barrierpenetration of said compounds.

WO 97/40049 describes several derivatives of benzazepines and relatedcompounds that may be applied for the treatment of Alzheimer's disease.However, no concept is provided in this application for increasing thepenetration of compounds through the blood-brain barrier.

SUMMARY OF THE INVENTION

Some embodiments provide compounds usable as pro-drugs or as amedicament having high pharmacodynamic effects in the brain's centralnervous system and low peripheral side effects.

Compounds disclosed herein include those described by formula (III):

-   -   wherein the bond <1> to >2> is a single or a double bond and the        bond between <3> and R1 is a single or a double bond and bond        <10> to <11> is a single or no bond and residues are        -   R1: OH, OCO-(3-pyridyl)(=nicotinic acid residue),            OCO-(3-methyl-3-pyridyl), OCO—(C₁-C₆ alkyl), OCO—(C₁-C₂₁            alkenyl), OCO—NH—(C₁-C₆ alkyl), OCO—(CH₂)_(x)—NH—COO—(C₁-C₆            alkyl), O—CH₂—O—(C₁-C₆ alkyl), O—(CH₂)_(x)—OCO—(C₁-C₆            alkyl), O—(CH₂)_(x)—OCO—(CH₂)_(x)—N—COO—(C₁-C₆ alkyl),            O—(CH₂)_(x)—OCO—(CH₂)_(y)-aryl, OCOO—(C₁-C₆ aminalkyl),            OCOO—(CH₂)_(x)-tetrahydrofuranyl, or a sugar, preferably            glucuronic acid residue, wherein x=1, 2, 3 or 4 and y=0, 1,            2, 3 or 4;            -   wherein if bond <3> to R1 is a double bond, then R1=O,                NH, NOH, NOR6, N—CO—NH₂, N—CS—NH₂, N—C(═NH)—NH₂,                N—NH-phenyl, N—NHR6, N—N(R6)₂, N—N═(CH₂)_(n), with                R6=C₁-C₅ unbranched or branched, saturated or                unsaturated (ar)alkyl, phenyl or benzyl and n=2-8; and            -   wherein if bond <3> to R1 is a single bond, then R1=OH,                SH, NH₂, NHR6, N(R6)₂, OR7, O—CR8R9, O—CO—CHR10,                NR11R12, or O—CO—R14,            -   with R7=C₁-C₂₂ unbranched or branched,                (poly-)unsaturated or saturated alkyl, optionally                containing an additional (ar)alkoxy or di(ar)alkylamino                group, a sugar or sugar derivative residue, preferably                glucuronic acid residue, a phosphoryl, alkylphosphoryl                or arylphosphoryl group, a sulfatyl or alkylsufatyl                group, or COR13,            -   where R13=R6 or R7 or pyridyl or dihydropyridyl or OR6,                preferably methyl, 3-pyridyl, 4-pyridyl,                3-dihydropyridyl, 4-dihydropyridyl            -   R8 and R9 are the same or different and any of H, Me, Ph                or they together form a spiro-ring —(CH₂)n- with n=4-6            -   R10=H or the side chain of a natural amino acid                including R10, R11 together are forming a proline or                hydroxy-proline derivative            -   R11 either is together with R10 forming a proline or                hydroxy-proline derivative or is H            -   R12 is a carbamate protecting group including                t-butoxycarbonyl, benzyloxycarbonyl and other                N-protecting groups;            -   R14 is an aromatic or heteroaromatic 5- or 6-membered                ring, selected from substituted benzene with the proviso                that it is not 2-fluorobenzene or                3-nitro-4-fluorobenzene, optionally substituted                naphthaline, thiophene, pyrrole, imidazole, pyrazole,                oxazole, thiazole; or CH(C₂H₅)CH₃, CH₂—C(CH₃)₃, or                cyclopropane;        -   R2: R7, or O—CR8R9, O—CO—CHR10, or NR11R12 with the same            definitions of R7-R12 as above, H, CH₃, CO—(C₁-C₆ alkyl),            CH₂—OCO—(CH₂)_(x)-aryl, or a sugar, preferably glucuronic            acid residue;        -   R3: H, F, Cl, Br, I, NH₂, NO₂, CN, CH₃;        -   R4: H, C₁-C₆ alkyl, preferably CH₃, CO—(C₁-C₆ alkyl),            CO-(3-pyridyl)(=nicotinic acid residue),            CO-(3-methyl-3-pyridyl),            CO—(CH-mercaptoalkyl)-(CH₂)_(x)-aryl,            (CH₂)_(x)—OCO—(CH₂)_(x)—N—COO—(C₁-C₆ alkyl),            (CH₂)_(x)—OCO—(CH-arylalkyl)-N—COO—(C₁-C₆ alkyl), wherein            x=1, 2, 3 or 4;        -   R5: if R4=H, then R5 is an electron pair;            -   if R4=CH₃ then R5 is an electron pair, hydrogen or a                C₁-C₅ (ar)alkyl group, CH₂—O—CH₃, CH₂—O—CO—R6,                CH₂—O—CR8R9, O—CO—CHR10, or NR11R12 with the same                definitions of R6 and R8-R12 as above, and wherein the                nitrogen at position <10> has an additional positive                charge as well as a counterion, selected from chloride,                bromide, iodide, sulphate, nitrate, hydrogensulfate,                phosphate, methanesulphonate, tosylate or any other                pharmaceutically acceptable anion, with the proviso that                the resulting compound is not Galantamine,                Norgalantamine, Sanguinine, Norsanguinine, Lycoramine,                Norlycoramine, Lycoraminone, Narwedine,                Nornarwedine,3-Amino-3-deoxy-galantamine or                3-amino-3-deoxy-1,2-dihydro-galantamine;            -   or R5=(CH₂)_(x)—O—(C₁-C₆ alkyl), (CH₂)_(x)—OCO—(C₁-C₆                alkyl), (CH₂)_(x)—OCO—(CH₂)-aryl,                (CH₂)_(x)—OCO—(CH₂)—N—COO—(C₁-C₆ alkyl), wherein x=1, 2,                3 or 4; wherein when bond <10> to <11> is a single bond                the nitrogen at position <10> has a positive charge and                the counterion is chloride, with the proviso that the                compound is not Galantamine, Norgalantamine, Sanguinine,                Norsanguinine, Lycoramine, Norlycoramine, Lycoraminone,                Narwedine, Nornarwedine,3-Amino-3-deoxy-galantamine or                3-amino-3-deoxy-1,2-dihydro-galantamine as a pro-drug or                medicament with improved blood-brain barrier                permeability compared to Galantamine.

Other embodiments relate to procedures for achieving a favorabledistribution ratio of brain to periphery for antidementia drugs ofvarious kinds, including cholinergic receptor sensitizing agents,cholinesterase inhibitors and neuroprotective drugs.

In this way the therapeutic effect-to-dose ratio can be increased andadverse side-effects can be reduced when the drugs are administered asmedicaments for the diseases mentioned in the present application. Thisobject is particularly met e.g., by site-specific chemical modification(derivatization) of said compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: 125 chemical structures and log P values of new compounds that(i) act as cholinergic enhancers, and/or (ii) have higher log P-valuesthan Galantamine (Galantamine included in Table 4 for comparison).

FIG. 2: Brain esterase inhibition by galantamine and severalpro-galantamines. A 20% mouse brain homogenate was used, supplementedwith 200 μM of acetylthiocholine as substrate, and the initial reactionkinetics were measured according to Riddles P W, Blakeley R L, ZernerB., “Reassessment of Ellman's reagent” Methods Enzymol. 1983, 91:49-60.As shown in the figure, even 50 μM of the respective pro-galantamineswere unable to achieve a level of inhibition of brain cholinesterasethat is comparable in size to that of 1 μM galantamine. A non-cleavablegalantamine derivative (Gln 1063) leads to negative values. Inderivative Gln 1063 R1 in Formula V is —O—Si(CH₃)₂—C(CH₃)₂—C(CH₃)₂H.

FIG. 3: Enzymatic cleavage of pro-galantamine to galantamine.Butyrylcholinesterase, 25 units/ml, was used. Reaction temperature was37° C. Appearance of the fluorescent reaction product galantamine wasdetermined by fluorescence detection.

FIG. 4: Interaction of galantamine and pro-galantamine with a4ß2neuronal nicotinic acetylcholine receptor ectopically expressed inHEK-293 cells. The increase in response to acetylcholine in the presenceof galantamine and Gln-1062, respectively, was determined by whole-cellpatch clamp recording. Galantamine achieved a maximal enhancement ofresponse of ˜40% whereas the pro-galantamine achieved a maximalenhancement of only ˜17%.

FIG. 5: Pharmacokinetics of pro-galantamine Gln-1062 (3 mg/kg) in themouse. (A) shows the measurable concentration of applied pro-galantamineand the resulting concentration of galantamine by cleavage of thepro-galantamine in brain and blood. The curve starting with the highestconcentration refers to derivative GLN-1062, which isbenzoyl-galantamine, in brain. (B) is an excerpt (“zoom”) of (A),showing the concentration range between 0.00 and 1.00 μg/g(substance/body weight) more in detail. In (B), the curve above refersto the concentration of resulting galantamine in brain, the middle curveshows the concentration of galantamine in blood and the curve startingwith a concentration of about 0.3 μg/g and decreasing refers to theconcentration of GLN-1062 in blood.

FIG. 6: Behavioral index for gastro-intestinal side effects in ferretsfollowing application of galantamine and several R1-pro-galantamines,respectively.

FIG. 7: Reversal from scopolamine-induced amnesia in mice, in thepresence of galantamine and several R1-pro-galantamines, respectively.Scopolamine induces a memory deficit that can be measured by increasedalternation in a T-maze trial. The cognition enhancing drugs were i.p.injected at several different doses together with scopolamine 20 minbefore a T-maze trial. Recovery was measured as a function of dose, andthe EC₅₀ was determined for each drug.

FIG. 8. Extraction Procedure. (A) Preparation of STD, QC and StudySamples. (B) Preparation of Calibration Curve Standards for Recovery.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present disclosure relates to significant enhancement in thebrain-to-plasma ratio of cholinergic receptor sensitizing agents, suchas the APL Galantamine (and related compounds), which is achieved byadministering not the drug itself but a “pro-drug” that is converted(back) to the drug itself inside the brain of the patient. As anothermeans for improving penetration through the blood-brain barrier (BBB)and thereby the therapeutic efficacy of the drug, the compoundsthemselves have been chemically modified so as to not only having largerefficacy as nicotinic APL and/or as neuroprotective agent, but inaddition having enhanced lipophilicity (higher log P) or otherwiseimproved BBB transport properties. Due to these improvements, thepro-drugs and other compounds addressed in this application should besignificantly more potent as medicaments for the treatment of cognitivedisorders than is, for example, Galantamine. The invention applies tothe compounds, selected pro-drugs and pharmaceutically acceptable saltsthereof, which might be administrated via the mouth, blood, skin, bynasal application, or any other suitable application route.

Herein the term “pro-drug” refers to a derivative of a base compoundwherein the group(s) added or replaced on said base compound are cleavedor returned to the group originally contained in the base compound whenthe derivative has reached the area or site of action. Thus, in case ofa “pro-drug”, an effective agent is administrated as a derivative (whichis said pro-drug), however, the compound mainly or exclusively effectiveat the target site within the brain is the agent itself, not thederivatized compound or metabolites thereof.

The term “derivative” refers to any change of a base compound defined inthe present application. The term “derivative” is used to describe acompound which either can be a pro-drug, or can be an effective agentitself/in its own right or in the derivatized form.

The terms “sensitizing agent” and “allosterically potentiating ligand,APL” refer to effectors that enhance cholinergic neurotransmission bydirect interaction via an allosteric site with cholinergic receptors.

The terms “cholinergic enhancer” and “cholinergic agent” refer tocompounds that enhance/modulate cholinergic neurotransmission byinhibition of cholinesterases, by allosteric sensitization and/or directactivation of cholinergic receptors and/or by activating/modulatingrelevant intracellular pathways via second messenger cascades.

A derivative or pro-drug has an “enhanced blood-brain barrierpermeability “according to the present invention or an “enhancedblood-brain barrier penetration” if, after administration of a pro-drugor derivative thereof to a living organism, a higher amount of saidcompound penetrates through the BBB, resulting in a higher level ofeffective agent in the brain, as compared to administration of the basecompound without derivatization. The enhanced BBB penetration shouldresult in an increased brain-to-tissue ratio of the effective agentcompared to the ratio of the base compound. Methods for determination ofan enhanced BBB permeability are disclosed in this application (seesupra).

The “base compound” according to the present invention preferably isGalantamine, Norgalantamine, Narwedine, N-Demethylnarwedine, Lycoramine,Lycoraminone, Sanguinine, Norsanguinine, and others (see table 1).

“log P” is defined as the decadic logarithm of the partition coefficientP which is the ratio of the concentration of a compound in aqueous phaseto the concentration of a compound in immiscible solvent, as the neutralmolecule.

The term “alkyl” shall mean a straight, branched or cyclic alkyl groupof the stated number of carbon atoms. Examples include, but are notlimited to methyl, ethyl, n-propyl, iso-propyl, n-butyl, isobutyl,sec-butyl, t-butyl, and straight and branched chain pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, pentadecyl etc. . . . orthe according cyclic alkyls.

The term “halo” shall mean chloro, fluoro, bromo and iodo.

The term “aryl” shall mean phenyl having 0, 1, 2 or 3 substituentsindependently selected from the group of alkyl, alkoxy, alkylcarbonyl,halo- or trihalomethyl.

The term “cycloalkyl” shall mean a cycloalkyl group of from 3 to 12carbon atoms and including multiple ring alkyls such as for example,adamantyl, camphoryl, and 3-noradamantyl.

In any case when a range between two limits is described it is meantthat any value or integer in this range is disclosed. For example“C₁-C₈” means C₁, C₂, C₃, C₄, C₅, C₆, C₇ or C₈; or “between 0,1 and 1”means 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9 or 1.

A “natural amino acid” is any amino acid naturally occurring inbiochemical pathways or in peptides/proteins. These are particularlyalanine, asparagine, cysteine, glutamine, phenylalanine, glycine,histidine, isoleucine, methionine, proline, glutamate, arginine, serine,threonine, valine, thryptophane, tyrosine, their methylated forms or theaccording salts.

With “sugar” is meant any suitable sugar, either an aldose or ketose, apyranose or furanose, heptose or hexose, mono- or polysaccharide, likee.g., glucuronic acid, glucose, fructose, galactose, mannose,saccharose, lactose, maltose etc., however, glucuronic acid ispreferred.

The main focus of the present invention is to improve blood-brainbarrier permeability, by increasing the lipophilicity or the transportproperties, or the ability of passing the blood-brain barrier, ofcompounds that are known to act as effective agents in correcting acholinergic deficit, e.g., APL of nicotinic receptors or inhibitors ofcholinesterases.

In one preferred embodiment the present invention refers to a method forincreasing blood-brain barrier penetration of a cholinergic enhancer bypreparing derivatives (either formally by their chemical structure ordirectly by chemical synthesis) of a molecule with a base structure ofthe general formula (I):

wherein the bond between positions <1> and <2> as well as <11> and <12>denotes a single- or double bond, and the bond between <10> and <11> iseither a single bond or no bond.

-   -   R1=═O, ═NOH, ═NH—NHCH₃, —OH, —OCOCH₃, —NH₂, or a (substituted)        derivative of the ketone, like semicarbazone, thiosemicarbazone,        aminoguanidine etc.    -   R2=H, CH₃, acetyl    -   R3=H, CH₃, F, Cl, Br, I    -   R4=H, CH₃.

In Table 1, compounds are exemplified with a base structure of thegeneral formula (II)

that belong to the structures summarized in formula (I):

TABLE 1 Bond Bond logP R1 R2 R3 R4 <1>—<2> <3>—R1 Name calcd.(1) OH CH₃H CH₃ Double Single Galantamine 1.30 OH CH₃ H H Double SingleNorgalantamine 1.38 OH H H CH₃ Double Single Sanguinine 0.83 OH H H HDouble Single Norsanguinine 0.91 MeCH(OH) H H CH₃ Double SingleLeucotamine 1.23 CH₂—CO OH CH₃ H CH₃ Single Single Lycoramine 1.28 OHCH₃ H H Single Single Norlycoramine 1.36 O CH₃ H CH₃ Single DoubleLycoraminone 0.85 O CH₃ H CH₃ Double Double Narwedine 0.74 O CH₃ H HDouble Double Nornarwedine 0.82 NH2 CH₃ H CH₃ Double Single3-Amino-3-deoxy- 1.05 galantamine NH2 CH₃ H CH₃ Single Single3-amino-3-deoxy- 0.89 1,2-dihydro- galantamine (1)Calculated usingAdvanced Pharma Algorithms Software ToxBoxes V1.0.2

The compounds listed in Table 1, and other compounds to be used as abase compound for derivatization according to the present invention, canbe obtained either by isolation from natural sources or by totalchemical synthesis, or by chemical modification of natural or syntheticcompounds.

The compounds to be used according to the present invention can bederivatives of the above listed molecules that can be demonstrated toact as cholinergic enhancers. This property of said derivatives may bemanifested by one or more of the following properties; by their abilityto sensitize cholinergic receptors, and/or inhibit braincholinesterases, and/or modulate intracellular messenger levels, and/oract neuroprotective. The ability to act as sensitizing agent onnicotinic receptors can be determined by electrophysiological andCa-imaging methods, as described in Schrattenholz A et al. (1996) MolPharmacol 49, 1-6 and Samochocki M et al (2000) Acta Neuro Scand Suppl176, 68-73; Samochocki M et al. (2003) J Pharmacol Exp Therap 305,1024-1036. The ability to inhibit cholinesterases can be determined bythe photometric method of Ellman et al., Biochem. Pharmacol. 7, 88(1961). The ability to modulate intracellular messenger levels can bedetermined by Ca-imaging methods (Samochocki M et al. (2003) J PharmacolExp Therap 305, 1024-1036) and other means of recording changes inintracellular messenger levels or effects resulting thereof (Kihara T etal (2004) Biochem Biophys Res Commun 325, 976-982). The ability to actneuroprotective can be determined by a variety of in vitro and in vivotest systems, including in cell culture (Arias E et al (2003)Neuropharmacol 46, 103-1S 14; Kihara T et al (2004) Biochem Biophys ResCommun 325, 976-982) and in animal models of neurodegenerative diseases(Capsoni et al (2002) Proc Natl Acad Sci USA 99, 12432-12437).

As specific examples, Table 2 exemplifies compounds that are derivativesof a base structure of the following general formula (III)

and act in any way as cholinergic enhancers:

TABLE 2 Bond Bond Bond Bond logP R1 R2 R3 R4 R5 1-2 3-R1 10-11 11-12Name calcd(1) OH CH₃ H CH₃ CH₃ D S n S 10,11-Seco- 2.6710-methyl-galantamine OH CH₃ H CH₃ H D S n D 10,11-Seco-11,12- 2.09dehydro-galantamine NOH CH₃ H CH₃ e D D S S Narwedinoxim 1.15 NNHCH₃ CH₃H CH₃ e D D S S Narwedin-N- 0.34 methyl-hydrazone OH CH₃ F CH₃ e D S S S8-Fluoro- 1.25 galantamine OH CH₃ Br CH₃ e D S S S 8-Bromo- 2.27galantamine OH CH₃ I CH₃ e D S S S 8-Iodo- 2.26 galantamine OH CH₃ BrCH₃ O D S S S 8-Bromo- 2.68 galantamine-N-oxide OH H H CH3 E D S S SSanguinine 0.83 O CH₃ H CH₃ e D D S S Narwedin 0.74 O CH₃ CH₃ CH₃ e D DS S 8-Methyl- 1.15 narwedine O—CO— CH₃ H CH₃ e D S S S GLN-0962 7.42(CH₂)11— CH₃ O—CO— CH₃ H CH₃ e D S S S GLN-0971 7.80 (CH2)6— CO—gal-6-yl O—CO— CO— H CH₃ e D S S S GLN-0935 11.7 (CH₂)₁₁— (CH₂)₁₁— CH₃ CH₃(1)Calculated using Advanced Pharma Algorithms Software ToxBoxes V1.0.2.Abbreviations: s: single bond; d: double bond; n: no bond; e: electronpair

Most of the compounds listed in Table 2 are not only efficacious agentsin one or more of the tests cited above, but most of them also have morefavourable log P and/or transport properties than the base compoundsfrom which they are derived.

To further improve BBB permeability and brain/plasma distribution ratio,modifications of the following kinds can be performed so as to make thecompounds exemplified in Tables 1 and 2 more lipophilic or enhanceotherwise their transport into the CNS, in comparison to the basecompound:

-   -   1. Conjugations to groups or molecules that are known to occur        in the course of metabolic conversions, e.g., carbohydrate        conjugates such as glycosyls, glucuronides and natural        metabolites, or are otherwise known to readily pass the        blood-brain barrier, e.g., amino acids, vitamins, various        messenger molecules and drugs.    -   2. Conjugations to groups leading to quaternary ammonium salts        with a labile nitrogen-carbon bond (see e.g., Example 1).    -   3. Conjugations to groups leading to esters, e.g., acylderivates        with enhanced lipophilicity and BBB penetration properties. For        example, such compounds may be esters of the oxygen function in        position 3 and/or 6 of the following base structure (IV):

-   -   -   a) Esters with saturated or unsaturated fatty acids            containing 1-22 carbon atoms optionally containing an            additional (ar)alkoxy or di(ar)alkylamino group        -   b) Esters with carbonic acid where one acidic function of            carbonic acid is esterified with the 3- and/or 6-position of            galantamine and the other represents an ester as defined in            3a.        -   c) Esters with (substituted) pyridine- or (substituted)            dihydropyridine-carboxylic acids (see e.g., Example 2)        -   d) Esters with phosphoric and sulfonic acids

    -   4. Formation of ketals or aminals of substituents in positions        3, 6, and 10 that increase the lipophilicity and are hydrolyzed        to the desired derivatives, e.g., (nor)galantamine derivatives        (see e.g., Examples 3 and 4)

    -   5. Formation of basic and/or quaternary carbamates of said        compounds that are chemically or metabolically unstable.

    -   6. Conjugation to a lipophilic dihydropyridinium carrier, e.g.,        as 1,4-dihydro-1-methyl-3-pyridinecarboxylate, that in the brain        is enzymatically oxidised to the corresponding ionic        pyrimidinium salt.

    -   7. Conjugation with nicotinic acid, nicotinic acid amide,        various cofactors, messenger molecules and other chemical        entities that enhance lipophilicity and transport through the        BBB.

These modifications lead to compounds of the following general formula(III)

wherein the bond between positions <1> and <2> denotes a single- ordouble bond, with the proviso that the structure is not any of thoselisted in Table 1 and the bonds <1> to <2> and <11> to <12> can beeither a single or a double bond, and the bond between <10> and <11> iseither a single bond or no bond and the residues R1-R5 are defined asfollows:R1:

-   -   a) if bond <3> to R1 is a double bond, then        -   R1=O, NH, NOH, NOR6, N—CO—NH₂, N—CS—NH₂, N—C(═NH)—NH₂,            N—NH-phenyl, N—NHR₆, N—N(R6)₂, N—N═(CH₂)_(n)        -   with R6=C₁-C₅ unbranched or branched, saturated or            unsaturated (ar)alkyl, phenyl or benzyl and n=2-8    -   b) if bond <3> to R1 is a single bond, then        -   R1=OH, SH, NH₂, NHR6, N(R6)₂, OR7, O—CR8R9, O—CO—CHR10, or            NR11R12            -   with R7=C₁-C₂₂ unbranched or branched,                (poly-)unsaturated or saturated alkyl, optionally                containing an additional (ar)alkoxy or di(ar)alkylamino                group, a sugar or sugar derivative residue, preferably                glucuronic acid, a phosphoryl, alkylphosphoryl or                arylphosphoryl group, a sulfatyl or alkylsufatyl group            -   or COR13,            -   where R13=R6 or R7 or pyridyl or dihydropyridyl or OR6,                preferably methyl, 3-pyridyl, 4-pyridyl,                3-dihydropyridyl, 4-dihydropyridyl            -   R8 and R9 are the same or different and any of H, Me, Ph                or they together form spiro-ring —(CH₂)_(n)— with n=4-6            -   R10=H or the side chain of a natural amino acid                including R10 and R11 together are forming a proline or                hydroxy-proline derivative            -   R11 either is together with R10 forming a proline or                hydroxy-proline derivative or is H            -   R12 is a carbamate protecting group including                t-butoxycarbonyl, benzyloxycarbonyl and other                N-protecting groups                R2:    -   H, R7, or O—CR8R9, O—CO—CHR10, or NR11R12, with the same        definitions of R7-R12 as above        R3:    -   H, F, Cl, Br, I, NH₂, NO₂, CN, CH₃        R4:    -   H or CH₃        R5:    -   If R4=H, then R5 is an electron pair    -   if R4=CH₃ then R5 is an electron pair, hydrogen or a C₁-C₅        (ar)alkyl group, CH₂—O—CH₃, CH₂—O—CO—R6, CH₂—O—CR8R9,        O—CO—CHR10, or NR11R12 with the same definitions of R6 and        R8-R12 as above.

In all the latter cases, the nitrogen bears an additional positivecharge as well as a counterion, selected from chloride, bromide, iodide,sulphate, nitrate, hydrogensulfate, phosphate, methanesulphonate,tosylate or other pharmaceutically acceptable anion.

Preferred derivatives of the main concept of the invention arequarternary ammonium salts with a labile nitrogen-carbon bond at R5;mono- or diacylderivatives (esters) of the hydroxyl groups of said basecompounds (R1, R2); sugar derivatives, preferably glucuronides (R1, R2);derivatives coupled with nicotinic acid (R1, R2); and selectedhalogenides (R3).

Another preferred derivative of the main concept is a lipophilicdihydropyridinium carrier. This Redox Chemical Delivery System (RCDS;Misra A et al (2003) J Pharm Pharmaceut Sci 6, 252-273) is known tosignificantly enhance drug delivery through the BBB into the brainparenchyma. Once inside the brain, the dihydropyridinium moiety isenzymatically oxidized to the corresponding ionic pyridinium salt.Subsequent cleavage of the original compound from the carrier leads toliberation of the original compound and to sustained levels of it in thebrain tissue.

Other preferred derivatives of the main concept are amino acids that areknown to be transported into the brain by active amino acid carriers,e.g., tyrosine. Once inside the brain parenchyma, these derivatives caneither directly act on their target molecules or are first enzymaticallyliberated before acting as the original aren't compound.

As a further aspect of the present invention, the derivatives obtainedby chemical modification do not need to work as such as medicaments butrather may initially be pro-drugs that, after penetration though theblood-brain barrier, are converted (e.g., by brain enzymes) to theparent compound or a metabolite thereof and work as such as amedicament. Said pro-drug or derivative is used to prepare a medicamentor pharmaceutical composition that preferably can be used for thetreatment of brain diseases associated with a cholinergic deficit.

Of the derivatives contained in the general structure of formula (III)and with the proviso and definitions provided there, the following areof particular interest in regard to the present invention, as they havenot yet been described or developed under the premise of having higherlipophilicity and/or better BBB transport properties and/or higherbrain-to-plasma ratio than their parent compounds (Table 1) from whichthey are derived by chemical modification:

TABLE 3 Examples of compounds described in previous publications/patentspresently shown that they (i) act as cholinergic enhancers, and/or (ii)have higher logP-values than Galantamine STRUCTURE logP Name

1.30 Galantamine

1.38 Nor- galantamine

1.68 3-O-Acetyl-6- O-demethyl- galantamine

1.72 8-Bromo- narwedine

1.76 Narcisine

1.99

2.15

2.27

2.35

2.69

3.07

3.27

4.09

4.90Han, So Yeop; Mayer, Scott C.; Schweiger, Edwin J.; Davis, Bonnie M.;Joullie, Madeleine M. Synthesis and biological activity of galantaminederivatives as acetylcholinesterase (AChE) inhibitors. Bioorganic &Medicinal Chemistry Letters (1991), 1(11), 579-80.

The following derivatives covered by the general structure of formula(III) and with the proviso and definitions provided there areparticularly preferred derivatives of the main concept of the inventionin that they have not yet been mentioned or described in any otherpublication or patent.

Examples of new compounds that (i) act as cholinergic enhancers, and/or(ii) have higher log P-values than Galantamine (the latter forcomparison only) are shown in FIG. 1.

The derivatives shown in Table 3 and FIG. 1 may be used to prepare amedicament or other pharmaceutical composition. Such medicament orpharmaceutical composition can be used for the treatment of a diseasestate associated with a cholinergic deficit.

The usefulness of the derivatives, before and/or after conversion to theparent compound, to act as effective pharmaceutical agents is manifestedby their ability to sensitize cholinergic receptors, and/or inhibitbrain cholinesterases, and/or modulate intracellular messenger levels,and/or act neuroprotective. The ability to act as sensitizing agent onnicotinic receptors can be determined by electrophysiological andCa-imaging methods, as described in Schrattenholz A et al. (1996) MolPharmacol 49, 1-6 and Samochocki M et al (2000) Acta Neuro Scand Suppl176, 68-73; Samochocki M et al. (2003) J Pharmacol Exp Therap 305,1024-1036. The ability to inhibit cholinesterases can be determined bythe photometric method of Ellman et al., Biochem. Pharmacol. 7, 88(1961). The ability to modulate intracellular messenger levels can bedetermined by Ca-imaging methods (Samochocki M et al. (2003) J PharmacolExp Therap 305, 1024-1036) and other means of recording changes inintracellular messenger levels or effects resulting thereof (Kihara T etal (2004) Biochem Biophys Res Commun 325, 976-982). The ability to actneuroprotective can be determined by a variety of in vitro and in vivotest systems, including in cell culture (Arias E et al (2003)Neuropharmacol 46, 103-1S 14; Kihara T et al (2004) Biochem Biophys ResCommun 325, 976-982) and in animal models of neurodegenerative diseases(Capsoni et al (2002) Proc Natl Acad Sci USA 99, 12432-12437).

This usefulness can also be ascertained by determining the ability ofthese compounds (1) to reduce neuronal cell death and amyloid plaqueformation as well as cognitive impairment in animal models ofAlzheimer's disease (Capsoni et al (2002) Proc Natl Acad Sci USA 99,12432-12437) and (2) to enhance learning performance in various animaltest systems. In one particular learning paradigm applied to old andyoung rabbits (Woodruff-Pak D et al (2001) Proc Natl Acad Sci USA, 98,2089-2094), the classical eye blink conditioning is used to study theeffect of cognition-enhancing drugs on the septohippocampal cholinergicsystem. An active test compound of the present invention will reduce thenumber of trials required to learn that the air blow applied onto theanimal's eye does not require the animal to close the eye (eye blink) asa protective measure.

This usefulness can also be ascertained by determining the ability ofthese compounds to restore deficient memory due to a cholinergic deficitin the Dark Avoidance Assay (DAA). In this assay mice are tested fortheir ability to remember an unpleasant stimulus for a period of e.g.,24 hours. A mouse is placed in a chamber that contains a darkcompartment; a strong incandescent light drives it to the darkcompartment, where an electric shock is administered through metalplates on the floor. The animal is removed from the testing apparatusand tested again, 24 hours later, for the ability to remember theelectric shock administered in the dark compartment.

If a nicotinic or muscarinic antagonist, i.e., an anticholinergic drugthat causes memory impairment, is administered before an animal'sinitial exposure to the test chamber, the animal tends to re-enter thedark compartment much sooner than in the absence of the anticholinergicdrug when being placed in the test chamber 24 hours later. This effectof an anticholinergic drug is blocked by an active test compound,resulting in a greater interval before re-entry into the darkcompartment.

The test results may be expressed as the percent of a group of animalsin which the effect of the anticholinergic drug is blocked or reduced,as manifested by an increased time interval between being placed in thetest chamber and re-entering the dark compartment.

According to the present intention and approach, the brain disease thatcan be treated with the pro-drugs and derivatives provided herewith canbe any psychiatric, neurological and neurodegenerative diseaseassociated with a cholinergic deficit of any kind, including aneurodegenerative loss of cholinergic neurotransmitters and/orreceptors, ACh-synthesizing and metabolizing enzymes, transport proteinsand the like. Such diseases are exemplified by Alzheimer's andParkinson's disease, other types of dementia, schizophrenia, epilepsy,stroke, poliomyelitis, neuritis, myopathy, oxygen and nutrientdeficiencies in the brain after hypoxia, anoxia, asphyxia, cardiacarrest, chronic fatique syndrome, various types of poisoning,anesthesia, particularly neuroleptic anesthesia, spinal cord disorders,inflammation, particularly central inflammatory disorders, postoperativedelirium and/or subsyndronal postoperative delirium, neuropathic pain,subsequences of the abuse of alcohol and drugs, addictive alcohol andnicotine craving, and subsequences of radiotherapy, and more. The effectof Galantamine or other cholinesterase inhibitors in treatment of suchdiseases are described e.g., in WO2005/74535, WO2005/72713,WO2005/41979, WO2005/30332, WO2005/27975, US2004/266659 andWO2004/14393.

All the derivatives described in the general (base) structure of formula(III) and Tables 2 and 3 and FIG. 1 either have an effect as a pro-drug,which means that the derivative, after entering the brain, is “convertedback” into an effective agent, e.g., Galantamine, Narwedine, Lycoramine,or the other said base compounds, or they are effective (i.e., ascholinergic enhancers or agents according to the definition) asderivatives themselves, meaning that they are not necessarily convertedor metabolised before they act as agents at their target molecules,e.g., cholinergic receptors or cholinesterases. The common feature ofthe derivatives of the present application is that they all penetratemore effectively through the blood-brain barrier than the base compound,which according to the present invention preferably is Galantamine andrelated compounds. As a result of their improved BBB penetrationproperties, these compounds should have higher therapeutic efficacy andlower adverse side effects than e.g., Galantamine.

The compounds of the present invention whether pro-drugs or otherwiseeffective agents can be administered as such or as a pharmaceuticallyacceptable salt thereof.

The derivatives of the common formulae as defined above can be preparedby any known method, however, it is preferred that the derivatives areprepared with proper use by the methods described for derivatization ofaccording compounds in EP-A 649 846 with reference to scheme I and inthe examples; EP-A648 771 with reference to scheme I and in theexamples; EP-A 653 427 with reference to scheme I and in the examples;U.S. Pat. No. 6,150,354, paragraph “procedures” and examples; or U.S.Pat. No. 6,638,925, paragraph “experimental section”, respectively. Afurther reference is WO 01/74820, wherein combinatory and/or parallelsynthesis is disclosed and synthesis of several compounds is describedin the examples. Further the method can be used as described in Gomes,P. et al., Rui. Centro de Investigacao em Quimica da Universidade doPorto, Oporto, Port. Synthetic Communications (2003), 33(10), 1683-1693.A skilled person clearly will understand that in any case an appropriateeduct/appropriate educts has/have to be used to obtain the desiredderivatization of the base structure. The preparation method is notlimiting the invention as long as the compounds presently described areobtained.

The compounds of the invention preferably are prepared from theappropriate optical isomer of Galantamine or Narwedine via theintermediate 6-demethylgalantamine, a known therapeutically effectivecompound, or 6-demethylnarwedine, respectively.

The pro-drugs and derivatives of this invention are selected by thefollowing tests, which shall be considered as examples not limiting theinvention:

-   -   1. Activity as nicotinic “allosterically potentiating ligand        (APL), preferentially determined by electrophysiological methods        and, Ca-imaging, using human cell lines that express individual        subtypes of human neuronal nicotinic acetylcholine receptors        (nAChR).        -   In the case of a compound acting as such: The activation of            nAChR by ACh or agonist is enhanced in the presence of said            compound, with the APL activity being selectively blocked by            antibody FK1.        -   In the case of a pro-drug: Enhanced activity as a centrally            acting APL after the pro-drug has been converted to the base            compound by treatment with a rat brain or human brain            homogenate extract.        -   Kinetics of conversion from pro-drug to drug when incubated            with a rat or human brain extract.    -   2. Activity as centrally acting cholinesterase inhibitor, as        tested by various in-vitro, cell culture and in-vivo test        systems.        -   In the case of a pro-drug: Enhanced cholinesterase            inhibition—or the same level of inhibition at a            significantly reduced dose—is observed when the pro-drug is            administered instead of the original base compound.        -   Kinetics of conversion from pro-drug to drug when incubated            with a rat or human brain extract.    -   3. Neuroprotective activity in acute toxicity protection tests        (organophosphate poisoning of animals, in-vitro poisoning by Aß        and/or glutamate) and in animal models of neurodegeneraton.        -   In the case of a pro-drug: Enhanced neuroprotective            activity—or the same level of neuroprotection at a            significantly reduced dose—is observed when the pro-drug is            administered instead of the original base compound.    -   4. Accumulation of the derivatives in the brain of mammals as        compared to unmodified Galantamine or other base compound.    -   5. Lipophilicity, as measured by shake-flask (e.g.,        octanol/buffer), HPLC-retention and nanobeads absorption        methods.    -   6. Bioconversion t_(1/2) in the brain as compared to blood        (systemic).    -   7. Theoretical/empirical estimates of distribution and log P        values.    -   8. Other miscellaneous tests.

As one way of estimating improved lipophilicity of the derivatizedcompounds, log P-values are provided in some of the tables. Improvedlipophilicity, as characterized by an increased log P-value, can eitherbe determined experimentally including HPLC methods or by predictivecomputational methods. Although such calculations cannot replace theexperiment, the data are strongly suggestive as to whether a certainmodification of the base compound will result in an improvedlipophilicity. Computer programs that allow such calculations includee.g., ToxBoxes from Pharma Algorithms, ACD-Lab, Molecule Evaluator fromCidrux, and others.

Another means of estimating the readiness of a compound to transversethe BBB is by experimental comparison of the membrane affinity of saidcompound to its binding affinity to serum albumin, both determined bythe NIMBUS Biotechnology assay (Willmann, S. et al. (2005) J Med Chem,in print).

Effective quantities of the compounds of the invention may beadministered to a patient by any of various methods, including orally asin capsules or tablets, via the skin or by nasal application. The freebase final products, while effective by themselves, may be formulatedand administered in the form of a pharmaceutically acceptable salt,e.g., for purposes of stability, convenience of crystallization,increased solubility, release retardation, and the like.

Since the pro-drugs/compounds of the present invention pass theblood-brain-barrier easier than the base compounds, there are twoadvantageous aspects: first is the fast uptake of the pro-drug andtherefore a fast onset of effect, second is that the dosage ofapplication can be decreased compared to known medicaments resulting inlower peripheral side effects with high efficacy of the compounds attheir effect site (brain). Further the pro-drugs after passage throughthe blood-brain-barrier are converted in the base compound which has alower permeability through the blood-brain-barrier, thus the effectivecompound remains in the brain, resulting in a longer time period ofeffectiveness.

As a representative case, the active compounds of the present inventionmay be orally administered, for example, with an inert diluent or withan edible carrier, or they may be enclosed in gelatine capsules, or theymay be compressed into tablets. Furthermore, the active compounds of theinvention may be incorporated with excipients and used in the form oftablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gum and the like. Preferred compositions and preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between 0.1 and 50 milligrams of active compound.

Because BBB penetration and brain-to-plasma ratio of the compoundsmodified according to this invention are significantly enhanced, thedosages of administered drug may be dramatically reduced, as compared toprevious applications, clinical studies and estimates.

Selected Aromatic and Heterocyclic Derivatives of Galantamine asPro-Drugs for the Treatment of Human Brain Diseases

The proposed derivatives were designed as pro-drugs, in the sense thatthey are able to effectively pass the blood brain barrier (BBB) and,after passing the BBB, they are substrates of endogenous enzymes and,upon enzymatic cleavage, produce galantamine. As a result of enzymaticcleavage to galantamine of such pro-galantamines in the brain, asignificantly higher local concentration of galantamine is achieved inthe brain than by administration of the same dose of originalgalantamine. The relatively higher drug concentration in the brainachieved by pro-galantamine administration will then result in higherefficacy at a given dose, and the better brain-to-peripheral tissuesdistribution will result in fewer or less significant side effects oftreatment. These effects are significant improvements of presenttreatment regimens because the efficacy as treatment for brain diseasesof unmodified galantamine (and all other ChE-I presently approved forthis purpose) is rather limited, albeit statistically significant,possibly due to low dosing. Thus, efficacy is usually reached only aftercareful (months-long) up-titration of daily dose, so as to maintainsufficient compliance of patients to the largely gastro-intestinal sideeffects associated with ChE-I treatment.

According to some embodiments, it was found that careful selectionconcerning the type of substituent and the position of substitutionusing galantamine as base structure result in highly efficaciouscompounds. Such very efficacious compounds having good blood brainbarrier passing properties and being efficiently cleaved by an esteraseafter passage through the BBB are obtained with compounds having thegeneral formula V

with R1 being a substituent having particular sterical and hydrophobicproperties.

These embodiments focus on esters of galantamine that, as such, havelittle or no activity as ChE-I and APL compared to galantamine.Therefore, as long as these compounds remain uncleaved, they do notinteract with the usual target molecules of galantamine and hence arelargely inactive in producing therapeutic and/or side effects. Thereduced reactivity of pro-galantamines is demonstrated by the followingresults:

-   -   1. Significantly reduced activity as ChE-I, as compared to        galantamine.    -   2. Reduced activity as nicotinic APL, as compared to        galantamine.    -   3. Reduced gastro-intestinal side effects, as compared to        galantamine.

All these approaches were investigated and are explained in the examplesand shown in the figures.

According to the invention it has been discovered that a particulargroup of esters of galantamine display unexpectedly high brain-to-bloodconcentration ratios (R_(BB)-proGal>6, as compared to R_(BB)-Gal ˜1.3)and in the brain they are relatively slowly enzymatically cleaved togalantamine. Therefore, as is discussed below in more detail, thesepro-galantamines are exceptionally well suited for the treatment ofhuman diseases associated with cholinergic deficits, such as Alzheimer'sdisease, Parkinson's disease, Schizophrenia and a variety of otherpsychiatric disorders.

The esters of galantamine to which the present invention refers to havethe following general structure:

wherein R1 either is CH(C₂H₅)CH₃, CH₂—C(CH₃)₃, or cyclopropane or beingan optionally substituted aromatic or hetero-aromatic 5- or 6-memberedring. Specifically, such aromatic and hetero-aromatic rings includebenzene, naphthalene, thiophene, pyrrole, imidazole, pyrazole, oxazoleand thiazole, in case that they are used as medicaments or pro-drugs forthe treatment of neurodegenerative or psychiatric or neurologicaldisease associated with a cholinergic deficit.

Such a disease preferably is selected from Alzheimer's and Parkinson'sdisease, other types of dementia, schizophrenia, epilepsy, neuritis,various types of poisoning, anesthesia, particularly neurolepticanesthesia, autism, spinal cord disorders, inflammation, particularlycentral inflammatory disorders, postoperative delirium and/orsubsyndromal postoperative delirium, neuropathic pain, subsequences ofthe abuse of alcohol and drugs, addictive alcohol and nicotine craving,and subsequences of radiotherapy.

The above mentioned compounds were not yet described for the treatmentof such diseases. Furthermore, compounds of formula I having aromatic orhetero-aromatic 5- or 6-membered ring, selected from substituted benzenewith the proviso that it is not 2-fluorobenzene or3-nitro-4-fluorobenzene, optionally substituted naphthalene, thiophene,pyrrole, imidazole, pyrazole, oxazole, thiazole; or CH(C₂H₅)CH₃,CH₂—C(CH₃)₃, or cyclopropane are according to the knowledge of theinventor not yet described at all.

In one preferred embodiment the compounds of the present invention areselected from

wherein R2-R6 comprising any substituent selected from H, halogen,optionally substituted C₁-C₃ alkyl or cyclopropyl, OH, O-alkyl, SH,S-alkyl, NH₂, NH-alkyl, N-dialkyl, optionally substituted aryl orhetero-aryl, whereby neighbouring substitutents can cooperate to form anadditional ring.

In another preferred embodiment of the present invention the compoundsare selected from the group consisting of the compounds as shown intable A, which is attached below.

Herein the term “pro-drug” refers to a derivative of galantamine (basecompound) wherein the group(s) added or replaced on said base compoundare cleaved or returned to the hydroxyl group originally contained inthe base compound, when the derivative has reached the area or site ofaction. Thus, in case of a “pro-drug”, an effective agent isadministrated as a derivative (which is said pro-drug), however, thecompound mainly or exclusively effective at the target site within thebrain is the agent itself, not the derivatized compound or metabolitesother than the base compound thereof.

The term “derivative” refers to any change of a base compound defined inthe present application. The term “derivative” is used to describe acompound which either can be a pro-drug, or can be an effective agentitself/in its own right or in the derivatized form.

The term “pro-galantamine” is used for any derivative of galantaminedescribed herein which can be cleaved by an enzyme (esterase) resultingin galantamine.

The terms “sensitizing agent” and “allosterically potentiating ligand,APL” refer to effectors that enhance cholinergic neurotransmission byinteraction with an allosteric site at cholinergic receptors.

The terms “cholinergic enhancer” and “cholinergic agent” refer tocompounds that enhance/modulate cholinergic neurotransmission byinhibition of cholinesterases, by allosteric sensitization and/or directactivation of cholinergic receptors and/or by activating/modulatingrelevant intracellular pathways via second messenger cascades.

A derivative or pro-drug has an “enhanced blood-brain barrierpermeability “according to the present invention or an “enhancedblood-brain barrier penetration” if, after administration of a pro-drugor derivative thereof to a living organism, a higher amount of saidcompound penetrates through the BBB of that organism.

A compound of the present invention provides an increased“brain-to-blood concentration ratio” or “brain-to-tissue concentrationratio” resulting in a higher level of effective agent in the brain, ascompared to administration of the base compound without derivatization.Methods for determination of an enhanced BBB permeability are disclosedin WO 2007/039138.

The “base compound” as well as the “effective agent” according to thepresent invention is galantamine. The effective agent is obtained by(local) enzymatic cleavage of the derivative.

“log P” is defined as the decadic logarithm of the partition coefficientP which is the ratio of the concentration of a compound in aqueous phaseto the concentration of a compound in immiscible solvent, as the neutralmolecule.

The term “alkyl” shall mean a straight, branched or cyclic alkyl group.As “alkyl” C₁ to C₁₀ alkyl groups are preferred, C₂ to C₈ groups aremore preferred and C₂ to C₆ groups are most preferred. C₁ to C₁₀ meansalkyl groups of the stated number of carbon atoms. Examples include, butare not limited to methyl, ethyl, n-propyl, iso-propyl, n-butyl,isobutyl, sec-butyl, t-butyl, and straight and branched chain pentyl,hexyl, heptyl, octyl, nonyl and decyl etc. . . . or the according cyclicalkyls.

The term “halo” shall mean chloro, fluoro, bromo and iodo.

The term “aryl” shall mean phenyl having 0, 1, 2, 3, 4 or 5 substituentsindependently selected from the group of alkyl, alkoxy, alkylcarbonyl,halo- or trihalomethyl.

The term “cycloalkyl” shall mean a cycloalkyl group of from 3 to 10carbon atoms and including multiple ring alkyls such as for example,adamantyl, camphoryl, and 3-noradamantyl.

In any case when a range between two limits is described it is meantthat any value or integer in this range is disclosed. For example“C₁-C₁₀” means C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀; or “between0,1 and 1” means 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9 or 1.

The stereo chemistry of the described derivatives are the same as thatof galantamine.

Benzoyl esters of galantamine were previously described in WO 9921561 A1Davis, Bonnie M. for a method of treatment of disorders of attentionwith galantamine, lycoramine, and related compounds, but no syntheses oranalytical or other data were provided for these compounds.

Substituted benzoyl esters were previously described in “Synthesis andbiological activity of galantamine derivatives as acetylcholinesterase(AChE) inhibitors” by Han, So Yeop; Mayer, Scott C.; Schweiger, EdwinJ.; Davis, Bonnie M.; Joullie, Madeleine M. Dep. Chem., Univ.Pennsylvania, Philadelphia, Pa., USA. Bioorganic & Medicinal ChemistryLetters (1991), 1(11), 579-80. CODEN: BMCLE8 ISSN: 0960-894X. Journalwritten in English. CAN 116:83569 AN 1992:83569 CAPLUS. In this documentthe synthesis of several ester and carbamate derivatives of galantamineare described as well as it was suggested that these compounds arepotential therapeutic agents in the treatment of Alzheimer's disease,based on their properties as AChE inhibitors.

In contrast to the teaching of these documents, the galantamine estersof the present invention have little, if any, activity asacetylcholinesterase inhibitors but rather are substrates of said enzyme(see above).

As representatively demonstrated for the benzoyl derivative in FIG. 2,these esters have little, if any cholinesterase inhibitory activity butrather are hydrolysed by cholinesterases to form galantamine andaccordingly act as pro-drugs of galantamine. As soon as galantamine isgenerated from these compounds, it acts as ChE-I and APL, as haspreviously been described. The structures of the tested derivatives canbe seen in Table 4. As a comparative derivative a non-cleavablegalantamine ether is also tested. Such derivative results in negativevalues of inhibition. In derivative Gln 1063 R1 in formula V is—O—Si(CH₃)₂—C(CH₃)₂—C(CH₃)₂H.

TABLE 4 Mol Reg. No. Molecular structure Abbreviation GLN-1062

Bz—Gal GLN-1081

4-Cl—Bz—Gal GLN-1082

4-MeO—Bz—Gal GLN-1083

4-Me—Bz—Gal GLN-1084

3,4-Cl2—Bz—Gal GLN-1085

4-tBu—Bz—Gal GLN-1086

3-CF3-4-Cl—Bz—Gal GLN-1088

4-CF3—Bz—Gal GLN-1089

2,4-Cl2—Bz—Gal GLN-1090

4-NO2—Bz—Gal GLN-1091

3-Cl—Bz—Gal GLN-1092

3-CF3—Bz—Gal GLN-1093

3-NO2—Bz—Gal GLN-1094

3,5-Cl2—Bz—Gal GLN-1095

3-Me2N—Bz—Gal GLN-1096

3-Me—Bz—Gal GLN-1097

2-Cl—Bz—Gal GLN-1098

2,4-F2—Bz—Gal GLN-1099

2,5-Cl2—Bz—Gal GLN-1100

4-F—Bz—Gal GLN-1101

4-NMe2—Bz—Gal GLN-1102

4-NH2—Bz—Gal GLN-1103

3-Me-4-NMe2—Bz—Gal GLN-1104

3,4-OCH2O—Bz—Gal GLN-1105

4-Ac—Bz—Gal GLN-1113

2-AcO—Bz—Gal GLN-0978

n-prop-Gal GLN-0979

i-but-Gal GLN-0992

GLN-0993

n-Hex-Gal GLN-1011

neo-pent-Gal GLN-1060

GLN-1061

GLN-1067

R/S-i-pent-Gal GLN-1069

GLN-1070

GLN-1071

GLN-1076

CyBu—Gal GLN-1077

GLN-1080

R/S-i-pent-Gal GLN-1106

3-Th—Bz—Gal GLN-1107

2-Th—Bz—Gal GLN-1108

5-Cl-2-Th—Bz—Gal GLN-1109

5-Im—Bz—Gal GLN-1110

5-OA—Bz—Gal GLN-1111

5-Th—Bz—Gal GLN-0926

Nic—Gal GLN-1066

Rather than inhibiting cholinesterases, the pro-galantamines referred toin the present document are substrates of the enzyme, as is exemplarilydemonstrated in FIG. 3.

The data of FIGS. 2 and 3 demonstrate that pro-galantamines of thepresent invention do not act as efficient inhibitors of cholinesterase,as was described in the earlier documents discussed above. Instead, theyare substrates of these enzymes. Similarly, they also do not interact tothe same extend as galantamine with neuronal nicotinic acetylcholinereceptors (FIG. 4).

The pro-galantamines of the present invention therefore either do notinteract, or only to a very limited extend, with the established targetmolecules of galantamine, in particular cholinesterases and neuronalnicotinic acetylcholine receptors. As pro-drugs they therefore haverather limited, if any, efficacy as cognition enhancers, and alsoproduce only limited peripheral and central side effects, as compared togalantamine (see further below).

As is exemplified and demonstrated by pharmacokinetics in mice (FIGS. 5aand 5b , Table 5), R1-benzoyl-galantamine displays an unexpectedly highbrain-to-blood concentration ratio (R_(BB)-proGal >19), a large initialconcentration in the brain, and it is only slowly cleaved togalantamine, as seen in the delayed appearance of a galantamine peak inbrain and blood. The R_(BB)-Value is significantly larger than what wasexpected from the log P value which probably is due to the slow cleavageof the pro-drug in the brain and a depot effect thereby produced.

In Table 5, the key pharmacokinetic data of benzoyl-galantamine, ofseveral other R1-pro-galantamines and of galantamine (for comparison)are listed.

TABLE 5 Pharmacokinetic data of several R1-pro-galantamines in the mouseGln number (for reference see table 4) logP Co (Brain) R-Pro R-Gal 10623.0 4812 19.3 2.2 1067 2.8 4665 7.5 1.7 0979 2.5 3166 6.2 79.4 0993 3.72150 6.4 0.6 0978 7.2 1985 6.9 2.3 1076 7.4 1245 1.1 1.0 Gal 1.7 17411.2Co is the highest pro-galantamine concentration (ng/ml) achieved inmouse brain after i.v. injection of 3 mg/kg of pro-galantamine. R-Proist he brain-to-blood concentration ratio of pro-galantamine, R-Gal thatof galantamine under these experimental conditions. For comparison, Coand R-Gal are also provided for i.v. injection of the same amount ofgalantamine.

These data establish that only a particular selection of R1substitutions is capable of producing the following advantageousproperties of pro-galantamines; high initial concentration in the brain,large R_(BB), and slow enzymatic conversion to galantamine. In addition(not shown in the table), the preferred R1-prodrugs display little, ifany side effects, as they are only very slowly converted to galantamine,thereby largely protecting them from acting as galantamine while beingtransported from the site of administration to the sites of action inthe brain.

These properties may have significant impact for the use of thesecompounds as drugs in Alzheimer's disease and other brain diseases. Asis representatively shown in FIG. 5, R1-pro-galantamines of the presentinvention display in ferrets much less gastro-intestinal side effectsthan galantamine. Ferrets were used in these studies as they are knownto be particularly sensitive to gastro-intestinal side effects. Inaddition to the classical emetic responses to galantamine and other ChEinhibitors, we recorded salivation (SA), shivering (SH), respiratoryproblems (RP) and diarrhea (DI) at the levels “none; 0”, “moderate; 0.5”(behaviour observed at low frequency and/or at low intensity) and“intense; 1.0” (behaviour observed frequently and/or continuously and/orat high intensity), and summated the scores for the four animals eachused per drug dose in these studies.

The data depicted in FIG. 6 for the two pro-galantamines demonstratethat their side effects profile in ferrets is much less severe (5-6times less) than that of the same dose of galantamine. The advantageousside effects profile is probably due to the reduced affinity ofinteraction of these R1-pro-galantamines with cholinesterases andneuronal nicotinic acetylcholine receptors (FIGS. 2, 4).

The advantages of enhanced transport of selected R1-produgs through theblood-brain barrier into the brain, enzymatic conversion to galantamineclose to target sites in the central nervous system, and interactionwith such sites is producing enhanced reversal of drug-induced amnesiain mice, as is shown in FIG. 7 for three pro-glantamines (and forgalantamine in comparison).

The data of FIG. 7 suggest that Gln-1062 is approximately 4-times morepotent than galantamine in reversing scopolamine-induced amnesia inmice. It may be expected that a similar or larger increase in drugefficacy can be achieved in man when the particular R1-pro-galantamineis administered instead of galantamine. The advantageous drug propertiesof R1-pro-galantamines (higher efficacy, lesser or less intense sideeffects) were also shown in other animal models.

In summary, the compounds of this invention are particularly useful asmedicaments for the treatment of human brain diseases associated with acholinergic deficit, including the neurodegenerative diseasesAlzheimer's and Parkinson's disease and the neurological/psychiatricdiseases vascular dementia, schizophrenia and epilepsy. Based onpreclinical studies using various animal models, the compounds havedramatically reduced side effects as compared to galantamine, includingmuch fewer, if any, incidents of emetic responses, diarrhea andvomiting. Moreover, when enzymatically cleaved, the resultinggalantamine displays an advantageous pharmacokinetic profile in thebrain and, due to its enhanced concentration level in the brain,displays also enhanced efficacy in interaction with the target moleculeslocated in the brain. Taken together, these properties make theadministration of galantamine as a R1-prodrug a preferred medication inthe diseases mentioned above.

Pharmaceutical Compositions and Administration

Acids useful for preparing the pharmaceutically acceptable acid additionsalts according to the invention include inorganic acids and organicacids, such as sulfamic, amidosulfonic, 1,2-ethanedisulfonic,2-ethylsuccinic, 2-hydroxyethanesulfonic, 3-hydroxynaphthoic, acetic,benzoic, benzenesulfonic acid, carboxylic, ethylenediamine tetraaceticacid, camphorsulfonic, citric, dodecylsulfonic, ethanesulfonic,ethenesulfonic, ethylenediamine tetraacetic, fumaric, glubionic,glucoheptonic, gluconic, glutamic, hexylresorcinic, hydrobromic,hydrochloric, isethionoc, (bi)carbonic, tartaric, hydriodic, lactic,lactobionic, laevulinic, laurylsulfuric, lipoic, malic, maleic, malonic,mandelic, methanesulfonic, mucic, naphthalenesulfonic, nitric, oxalic,pamoic, pantothenic, perchloric, phosphoric, polygalacturonic, pectic,propionic, salicylic, succinic or sulfuric acid, p-tuluenesulfonic,wherein hydrochloric, hydrobromic, sulfuric, nitric, phosphoric andperchloric acids, as well as tartaric, citric, acetic, succinic, maleic,fumaric and oxalic acids are preferred.

The active compounds of the present invention may be orallyadministered, for example, with an inert diluent or with an ediblecarrier, or they may be enclosed in gelatin capsules, or they may becompressed into tablets. For the purpose of oral therapeuticadministration, the active compounds of the invention may beincorporated with excipients and used in the form of tablets, troches,capsules, elixirs, suspensions, syrups, wafers, chewing gum and thelike. These preparations should contain at least 0.5% of activecompounds, but may be varied depending upon the particular form and mayconveniently be between 5% to about 70% of the weight of the unit. Theamount of active compound in such compositions is such that a suitabledosage will be obtained. Preferred compositions and preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between 0.1-50 milligrams of active compound.

The tablets, pills, capsules, troches and the like may also contain thefollowing ingredients: a binder such as micro-crystalline cellulose, gumtragacanth or gelatin: an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, cornstarch and thelike; a lubricant such as magnesium stearate or Sterotex; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin may be added or a flavouring agent such as peppermint, methylsalicylate, or orange flavouring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above-type, aliquid carrier such as an oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus, tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes, colourings and flavours. Materials used inpreparing these various compositions should be pharmaceutically pure andnon-toxic in the amounts used.

For the purpose of nasal or parenteral therapeutic administration, theactive compounds of the invention may be incorporated into a solution orsuspension. These preparations should contain at least 0.1% of activecompound, but may be varied between 0.5 and about 30% of the weightthereof. The amount of active compound in such compositions is such thata suitable dosage will be obtained. Preferred compositions andpreparations according to the present inventions are prepared so that anasal or parenteral dosage unit contains between 0.1 to 20 milligrams ofactive compound.

Further the compounds of the present invention can be administered viaintranasal delivery to the cerebral spinal fluid as disclosed in detailin WO2004/02404.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents, such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylene-diamine tetraacetic acid; buffers suchas acetates; citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. Parenteral multiple dosevials may be of glass or plastic.

Typical dosage rates in administration of the active ingredients dependon the nature of the compound that is used and in intravenousadministration are in the range of 0.01 to 2.0 mg per day and perkilogram of body weight based on the physical condition and othermedications of the patient.

The following specific formulations exemplify suitable applications:Tablets and capsules that contain 0.5 to 50 mg. Solution for parenteraladministration that contains 0.1 to 30 mg of active ingredient/ml.Liquid formulations for oral administration at a concentration of 0.1 to15 mg/ml. Liquid formulations for nasal or intra-cerebroventricularadministration at a concentration of 0.1 to 5 mg of activeingredient/ml. The compounds according to the invention can also beadministered by a transdermal system, in which 0.1 to 10 mg/day isreleased. A transdermal dosage system may consists of a storage layerthat contains 0.1 to 30 mg of the active substance as a free base orsalt, in case together with a penetration accelerator, e.g., dimethylsulfoxide, or a carboxylic acid, e.g., octanoic acid, and arealistic-looking polyacrylate, e.g., hexylacrylate/vinylacetate/acrylic acid copolymer including softeners, e.g.,isopropylmyristate. As a covering, an active ingredient-impermeableoutside layer, e.g., a metal-coated, siliconised polyethylene patch witha thickness of, for example, 0.35 mm, can be used. To produce anadhesive layer, e.g., a dimethylamino-methacrylate/methacrylatecopolymer in an organic solvent can be used.

The invention also relates to pharmaceutical compositions that in apharmaceutically acceptable adjuvant contain a therapeutically effectiveamount of at least one of the compounds that are proposed according tothe invention.

Examples of chemical synthesis and properties of derivatives are givenin the following examples. Abbreviations: DCM: dichloromethane; DMAP:4-dimethylaminopyridine; DCC: dicyclohexylcarbodiimide; DCHU:dicyclohexylurea.

Example 1 N-Methoxymethyl-galanthaminiumchloride(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-11-methoxymethyl-11-methyl-6H-6-hydroxy-3-methoxy-benzofuro[3a,3,2-ef][2]benzaze-pinium,chloride)

N-Methoxymethyl-galanthaminiumchloride is obtained from Galantamine viaalkylation using chloromethylmethylether:

To a solution of (−)-Galantamine (5.00 g, 17.4 mmol) in drydimethylformamide (12 mL) chloromethylmethylether (1.12 g, 13.9 mmol) isadded at −5 bis 0° C. in the course of 15 min and stirred for 4 hrs. atroom temperature. The reaction mixture is poured on ethyl acetatet (500mL) and the precipitate obtained is filtered and washed using ethylacetate (3×50 mL).

The crude product (4.20 g, 82%) has a purity of 96% (HPLC). For furtherpurification the crude product is dissolved in dry ethanol, stirredafter the addition of activated charcoal, filtered and added to ethylacetate (500 mL). The precipitate is filtered and washed using ethylacetate (3×50 mL) and dry diethylether (1×50 mL). The product isobtained in the form of colourless crystals (3.85 g, 75% d. Th.) meltingat 126-127° C.

Opt. Rotation: [α]_(D) ²⁰=−113.9° (c=0.18 g/water) calcd. ForC₁₉H₂₆ClNO₄*0.33H₂O C, 61.04; H, 7.19; N, 3.75. found: C, 61.10; H,7.07; N, 3.75.

¹H NMR (DMSO-d6) δ 6.86 (s, 2H), 6.29 (d, J=10 Hz, 1H), 5.88 (d, J=10Hz, J=4 Hz, 1H), 5.13 (bs, 3H), 4.66 (s, 2H), 4.48 (d, J=14 Hz, 1H),4.22-3.90 (m, 2H), 3.81 (s, 3H), 3.70 (s, 3H), 3.70-3.52 (m, 1H), 2.75(s, 3H), 2.44-1.79 (m, 4H); 13C NMR (DMSO-d6) δ 146.4 (s), 145.2 (s),132.8 (s), 130.2 (d), 125.3 (d), 123.7 (d), 117.8 (s), 112.1 (d), 94.8(t), 86.4 (d), 61.7 (d), 60.3 (t), 59.4 (q), 56.2 (t), 55.6 (q), 46.2(s), 40.2 (q), 31.1 (2 t);

The chemical and biological stability of this compound has beendetermined in various buffers (chemical stability), in rat blood serum,and in rat brain extract, suggesting that the derivative can act as apro-drug.

Instead of chloromethyl or methyl ether the following reagents can beused alternatively: Methoxymethanolbenzenesulfonate,trifluoromethanesulfonic acid methoxymethyl ester, or methoxymethanol4-methylbenzenesulfonate.

Example 2 Tert-Butoxycarbonylamino-acetic acid(N-norgalanthaminyl)-methyl ester

To a solution of N-Boc-glycine chloromethylester (1.0 mmol) andnorgalantamine (1.0 mmol) in dry DMF (2.0 mL) triethylamine (3 mmol) wasadded dropwise and the reaction stirred under nitrogen for 3 days. Thetriethylammonium chloride formed was filtered and washed with dry etherand the filtrate rotoevaporated to dryness. The residue was redissolvedin dry acetone (2 ml) upon heating and left to stand overnight at 4° C.for additional precipitation of the triethylammonium salt. After renewedfiltration and rotoevaporation the mixture was chromatographed on silicausing ethyl acetate/petrol ether. The target product was isolated as anoil.

¹³H NMR (DMSO-d6) δ 28.5, 33.9, 37.9, 42.0, 48.2, 51.2, 56.2, 56.9,61.9, 79.5, 79.9, 88.8, 111.8, 121.3, 126.6, 129.8, 130.8, 133.6, 145.8,148.2, 156.3, 169.6.

Example 3 2-tert-Butoxycarbonylamino-3-phenylpropionic acetic acid(N-norgalantha-minyl)-methyl ester

This compound was prepared using the procedure of example 2 withN-Boc-phenylalanine chloromethylester.

¹³H NMR (DMSO-d6) δ 28.5, 33.9, 36.9, 37.9, 48.2, 51.2, 54.6, 56.2,56.9, 61.9, 79.5, 80.2, 88.8, 111.8, 121.3, 126.0, 126.6, 127.8, 128.7,129.8, 130.8, 133.6, 139.5, 145.8, 148.2, 156.0, 171.6.

Example 4(3R,4aS,9bS)-9-Dimethylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-9b-vinyl-dibenzofuran-3-ol;(=10,11-Seco-11,12-dehydro-10-methyl-galantamine)

A solution of N-methylgalanthaminium iodide (5.0 g, 11.6 mmol) in 35%aqueous potassium hydroxide (150 mL) is heated under reflux for 48 hrs,diluted with water (200 mL) and acidified using conc. hydrochloric acidto pH=3-4 and extracted with dichloromethane (2×50 mL) to removenon-basic compounds. The aqueous phase is basified using cone. ammoniato pH 12 and extracted using dichloromethane (4×100 mL). The combinedorganic extracts are washed with brine (2×50 mL), dried using sodiumsulfate and rotoevaporated to obtain the crude product which is purifiedby MPLC (200 g SiO₂, chloroform:methanol=99:1+1% conc. ammonia). Theproduct is obtained as yellow oil (2.5 g, 71% d. Th.). The fumarate(colourless crystals) and oxalate salt (off-white crystals) whereobtained in the usual way: m.p.: 151-153° C. (fumarate), 116-118° C.(oxalate). [α]_(D) ²⁰=−56.5° (0.212 g/100 mL H₂O) (fumarate).

fumarate: oxalate C₁₈H₂₅NO₃ * 1.0C₄H₄O₄ C₁₈H₂₅NO₃ * 1.0C₂H₂O₄ * 0.75H₂OCalcd.: C, 62.99; H, 6.97; N, 3.34 Calcd.: C, 59.32; H, 6.60; N, 3.46Found: C, 62.89; H, 6.62; N, 3.32 Found.: C, 59.48; H, 6.31; N, 3.38

¹H-NMR (CDCl3): δ 6.83 (d, J=8.4 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H),6.13-5.95 (m, 3H), 5.32 (dd, J=10.3, 1.1 Hz, 1H), 5.25 (dd, J=18.3, 1.1Hz), 4.63 (b, 1H), 4.15 (b, 1H), 3.85 (s, 3H), 3.58 (d, J=12.8 Hz, 1H),3.07 (d, J=12.8 Hz, 1H), 2.56 (m, 1H), 2.15 (s, 6H), 1.96 (ddd, J=16.2,4.9, 2.3 Hz, 1H); ¹³C-NMR (CDCl3): δ 146.6 (s), 144.1 (s), 139.0 (t),132.2 (s), 128.6 (s), 128.1 (d), 127.8 (d), 123.6 (d), 117.3 (t), 111.1(d), 86.0 (d), 62.0 (t), 59.7 (t), 55.7 (q), 52.9 (s), 44.7 (q), 28.6(t)

Example 5(3R,4aS,9bS)-6-Methoxy-9-methylaminomethyl-3,4,4a,9b-tetrahydro-9b-vinyl-dibenzofuran-3-ol;(=10,11-Seco-11,12-dehydro-galantamine)

3-Chloroperbenzoic acid (0.38 g, 75% ig, 1.66 mmol) is added to asolution of(3R,4aS,9bS)-9-dimethylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-9b-vinyl-dibenzofuran-3-ol(0.50 g, 1.66 mmol) in dichloromethane (35 mL) and then stirred for 30minutes at room temperature. After adding a solution ofiron(II)sulfate-heptahydrate (0.23 g, 0.83 mmol) in methanol (5 mL) itis then stirred for another 20 minutes at room temperature. Then 2Nhydrochloric acid (30 mL) is added, stirred for 5 minutes and most ofthe dichloromethane is removed by rotoevaporation. The remaining aqueousphase is washed with diethyl ether (4×20 mL), basified to pH 12 usingconcentrated ammonia and then extracted with dichloromethane (4×40 mL).The combined organic phases are washed with saturated sodium chloridesolution (30 mL), dried using sodium sulphate, filtered and the solventis again removed by rotoevaporation to obtain the crude product which isthen further purified using MPLC (Büchi, 110 g SiO₂, chloroform:methanol97:3+1% concentrated ammonia) and obtained as a yellow oil (0.30 g, 63%d. Th.). The oxalate is prepared in the usual way and obtained ascolourless crystals, 0.37 g, 59% d. Th., m.p. 127-129°. The purity ischecked by TLC (chloroform:methanol=9:1+1% conc. ammonia, Rf=0.35).[α]_(D) ²⁰=−41.8° (0.220 g/100 mL H₂O) (Oxalate)

C₁₇H₂₁NO₃*1.0C₂H₂O₄*0.5H₂O

Calcd.: C, 59.06; H, 6.26; N, 3.62.

Found: C, 59.35; H, 6.00; N, 3.56.

¹H-NMR (CDCl3): δ 6.88 (d, J=8.4 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H),6.15-5.82 (m, 3H), 4.67 (b, 1H), 4.09-4.20 (m, 1H), 3.85 (s, 3H), 3.68(s, 2H), 2.52-2.49 (m, 1H), 2.42 (s, 3H), 1.97 (ddd, J=16.2, 4.9, 2.3Hz, 1H); 13C-NMR (CDCl3): δ 146.6 (s), 144.0 (s), 139.4 (d), 131.6 (s),129.5 (s), 128.9 (d), 127.2 (d), 122.7 (d), 117.6 (t), 111.8 (d), 86.0(d), 62.0 (d), 55.9 (q), 52.8 (t), 51.1 (s), 36.0 (q), 28.8 (t)

Example 6(3R,4aS,9bS)-9-Dimethylaminomethyl-9b-ethyl-6-methoxy-3,4,4a,9b-tetrahydro-dibenzofuran-3-ol;(=10,11-Seco-10-methyl-galantamine)

Palladium (10%) on active carbon (90 mg) is pre-hydrogenated in methanol(40 mL) and conc. acetic acid (2 mL) in the Parr-apparatus at 10 psi androom temperature for 45 minutes. After adding(3R,4aS,9bS)-9-dimethylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-9b-vinyl-dibenzofuran-3-ol(0.90 g, 2.99 mmol) it is then hydrated for 8 hrs. at 15-20 psi and roomtemperature. The catalyst is then filtered and the solvent is removed byrotoevaporation. The residue is then dissolved in water (100 mL),basified using conc. ammonia and extracted using dichloromethane (5×40mL). The combined aqueous phases are washed with a saturated sodiumchloride solution (2×20 mL), dried using sodium sulphate and the solventis removed by rotoevaporation. It is then further purified using MPLC(Büchi, 110 g SiO₂, chloroform:methanol=98:2+1% conc. ammonia), obtainedas a colourless oil (0.80 g, 88%) and converted to the hydrochloridem.p. 248-249°. [α]_(D) ²⁰=−47.3° (0.220 g/100 mL H₂O). TLCchloroform:methanol=9:1+1% conc. ammonia, Rf=0.45.

C₁₈H₂₅NO₃*2.0 HCl

Calcd.: C, 57.45; H, 7.23; N, 3.72.

Found: C, 57.95; H, 6.85; N, 3.48.

¹H-NMR (CDCl3): δ 6.78 (d, J=8.3 Hz, 1H), 6.67 (d, J=8.3 Hz, 1H), 6.12(d, J=10.2 Hz, 1H), 5.89 (dd, J=10.2, 4.3 Hz, 1H), 4.74 (b, 1H),4.19-4.09 (m, 1H), 3.84 (s, 3H), 3.54 (d, J=12.9 Hz, 1H), 3.19 (d,J=12.9 Hz, 1H), 2.53-2.32 (m, 1H), 1.94-2.13 (m, 2H), 1.69 (ddd, J=16.2,4.9, 2.3 Hz, 1H), 0.85 (t, J=7.6 Hz, 3H); ¹³C-NMR (CDCl3): δ 146.8 (s),144.4 (s), 131.6 (d), 128.2 (s), 127.7 (d), 123.6 (d), 110.6 (d), 83.8(d), 62.7 (d), 61.6 (s), 55.7 (q), 51.1 (s), 45.2 (q), 31.6 (t), 27.5(t),

Example 7 3.3.4.(3R,4aS,9bS)-9b-Ethyl-9-methylaminomethyl-6-methoxy-3,4,4a,9b-tetrahydro-dibenzofuran-3-ol;(=10,11-Seco-galantamine)

Following the procedure of example 6 using(3R,4aS,9bS)-9-Dimethylaminomethyl-9b-ethyl-6-methoxy-3,4,4a,9b-tetrahydro-dibenzofuran-3-olthe pure product is obtained as a yellow oil (0.17 g, 59% d. Th.) andconverted to the oxalate and fumarate.

M.p. (oxalate) 162-164°, [α]_(D) ²⁰=−51.2° (0.146 g/100 mL H₂O)(oxalate).

TLC chloroform:Methanol=9:1+1% conc. ammonia, Rf=0.39

fumarate: oxalate C₁₇H₂₃NO₃ * 1C₄H₄O₄ * 0.33H₂O C₁₇H₂₃NO₃ * 1C₂H₂O₄ *0.25H₂O Calcd.: C, 61.31; H, 6.78; N, 3.40 Calcd.: C, 59.44; H, 6.69; N,3.65 Found: C, 61.22; H, 6.67; N, 3.22 Found; C, 59.53; H, 6.78; N, 3.65

¹H-NMR (CDCl3): δ 6.85 (d, J=8.4 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H),5.97-5.92 (m, 2H), 4.74 (dd, J=5.8, 3.5 Hz, 1H), 4.22-4.12 (m, 1H), 3.84(s, 3H), 3.74 (d, J=7.2 Hz, 2H), 2.48 (s, 3H), 2.45-2.28 (m, 2H),2.20-1.62 (m, 5H), 0.85 (t, J=7.46, 3H); ¹³C-NMR (CDCl3): δ 146.6 (s),144.2 (s), 131.1 (s), 131.0 (d), 128.9 (s), 128.8 (d), 122.3 (d), 111.1(d), 83.8 (d), 62.8 (d), 55.8 (q), 51.0 (s), 36.3 (q), 32.5 (t), 29.1(t),

Example 8(4aS,6R,8as)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-yl-β-D-glucopyranosiduronicacid (=Galantamine-3-glucuronide)

Step 1: Methyl 1,2,3,4-tetra-O-isobutyryl-β-D-glucopyranuronate (2)

To a solution of NaOMe (26 mg, 0.48 mmol) in MeOH (150 mL) was addedglucurono-6,3-lactone (20.6 g, 154 mmol) in portions with stirring untildissolved. The solvent was then removed in vacuo, the residue taken upin pyridine (85 mL, 1.08 mol) and the solution cooled to 0° C.Isobutyryl chloride (110 mL, 1.06 mol) in CH₂Cl₂ (70 mL) was then addedwith strong mechanical stirring at a rate that kept the temperaturebelow 10° C., and the reaction mixture was left at room temperatureovernight. More CH₂Cl₂ (100 mL) was then added and the solution washedwith water (400 mL), 2 M HCl (3×50 mL), saturated sodium bicarbonate(5×50 mL) and brine (50 mL). After drying, filtering and evaporating invacuo, a gum was obtained which crystallized on trituration withpetroleum ether (40-60° C.). Filtration and drying at 40° C. in a vacuumoven yielded the title product. Recrystallisation from MeOH or petrolether afforded the pure β isomer 2 as needles, mp 127° C., (21.6 g, 37%,from mother liquid some more product could be isolated) [α]_(D)=+11.12(c 1.7 CHCl3); δ_(H) (300 MHz, CDCl3): 5.78 (d, J=8 Hz), 5.39 (t, J=9.5Hz), 5.25 (t, J=9.5 Hz), 5.23 (dd, J=9.5, 8 Hz), 4.19 (d, J=9.5 Hz),3.75(s, OMe), 2.65-2.45 (m, 4×CHMe₂), 1.17-1.07 (m, 4×CHMe₂).

An alternative procedure with pivaloyl chloride was also used to preparemethyl 1,2,3,4-tetra-O-pivaloyl-β-D-glucopyranuronate in 21% (theisolation and crystallization of compound 2 was easier).

Step 2: Methyl 2,3,4-tri-O-isobutyryl-D-glucopyranuronate (3)

Ammonia gas pre-dried by passing it through a bed of sodium hydroxidewas bubbled through CH₂Cl₂ (200 mL) at −4° C. over 1 h at a rate whichkept the temperature below 0° C. The above methyl1,2,3,4-tetra-O-isobutyryl-β-D-glucopyranuronate (3.0 g, 8 mmol) wasadded and the solution stirred at 0° C. for 3 h and then left at roomtemperature for 20 h. Nitrogen gas was bubbled through the solution for30 min. and it was extracted with ice-cold 10% aqueous HCl, then water.The organic phase was dried over Na₂SO₄ filtered and solvent removed invacuo to leave the crude product. Recrystallization from CHCl₃:PEafforded the pure microcrystalline α-epimer, mp 89° C. δH (300 MHz,CDCl3): 5.65 (t, J=10 Hz), 5.54 (d, J=3.5 Hz), 4.92 (dd, J=10, 3.5 Hz),4.60 (d, J=10 Hz), 3.75 (s, OMe), 2.61-2.43 (m, 4×CHMe₂), 1.20-1.05 (m,4×CHMe₂).

Step 3: Methyl2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-D-glucopyranuronate(4)

To a stirred solution of methyl2,3,4-tri-O-isobutyryl-D-glucopyranuronate 3 (418 g, 1 mmol) in CH₂Cl₂(5 mL) was added trichloroacetonitrile (0.4 mL, 3.7 mmol), followed byanhydrous potassium carbonate (83 mg, 0.6 mmol), and the mixture stirredfor 40 h. It was filtered through a short pad of silica and eluted withether. Filtration and evaporation in vacuo then yielded the titleproduct 4 as a semi crystalline gum which crystallized from dryisopropanol as white prisms, mp 108° C. (422 mg, 75%). δH (300 MHz,CDCl3): 8.72 (s, NH), 6.66 (d, J=3.5 Hz), 5.70 (t, J=10 Hz), 5.30 70 (t,J=10 Hz), 5.20 (dd, J=10, 3.5 Hz), 4.51 (d, J=10 Hz), 3.75 (s, OMe),2.60-2.43 (m, 3×CHMe₂), 1.17-1.06 (m, 3×CHMe₂).

Step 4: Galantamine-6-methyl2,3,4-tri-O-isobutyryl-β-D-glucopyranuronate (5)

A suspension of dried galantamine hydrobromide (92 mg, 0.25 mmol) andthe above methyl2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-β-D-glucopyranuronate 4(282 mg, 0.5 mmol) in dry CH₂Cl₂ (10 mL) containing 4 Å molecular sieveswas stirred under argon at room temperature, while BF₃.Et₂O (0.1 mL, 0.5mmol) was added. After 1 h, virtually all of the starting materials haddissolved and stirring was continued for 2 days. More CH₂Cl₂ (20 mL) wasadded, the solution washed with saturated aq. sodium bicarbonate (10mL), water and brine before being dried. Filtration and evaporation invacuo afforded a semisolid residue, which was purified with MPLC onsilica. Elution with CHCl₃/MeOH 97:3-20 gave 75 mg of the glucuronide.Trituration with EtOH yielded 30 mg of pure 5.

Step 5:(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-yl-β-D-glucopyranosiduronicacid (Galantamine-3-glucuronide) (6)

2M-NaOH (2.0 mL) was added to a stirred suspension of the glucuronate 5(30 mg) in MeOH (4 mL), and the mixture left overnight. The solution wasthen acidified with glacial acetic acid to pH 5.5, the solventevaporated and purified over silica with CHCl₃: MeOH (saturated with dryNH₃) 95:5. The product-fraction was freeze-dried to afford 14 mg of 6 asa white powder, m.p. 238° (dec.).

¹H NMR (MeOD, 200 MHz): 1.63-1.73 (m, 1H), 2.02-2.21 (m, 2H), 2.38 (s,3H), 2.43-2.53 (m, 1H), 2.99-3.06 (m, 1H), 3.19-3.33 (m, 1H), 3.47-3.49(m, 1H), 3.65-3.71 (d, 1H, J=14.9 Hz), 3.78 (s, 3H), 4.05-4.13 (d, 1H,J=14.9 Hz), 4.58 (m, 1H), 5.85-5.94 (dd, 1H, J2=4.8 Hz, J2=10.2 Hz),6.15-6.21 (d, 1H, J2=10.2 Hz), 6.63-6.77 (m, 2H)

¹³C NMR (MeOD, 200 MHz): 23.22, 28.65, 34.57, 42.02, 43.33, 48.09,54.03, 55.64, 60.43, 88.54, 112.18, 122.30, 127.16, 127.70, 128.64,133.49, 144.65, 146.39

Example 9 Galantamine-3,6-di-β-D-glucuronide

Step 1: Galantamine-3,6-di(methyl2,3,4-tri-O-isobutyryl-3-D-glucopyranuronate) (7)

Following the procedure for the preparation of Galantamine-6-methyl2,3,4-tri-O-isobutyryl-β-D-glucopyranuronate but using sanguinine (137mg, 0.5 mmol) and the above imidate 4 (1.12 g, 2 mmol) in dry CH₂Cl₂ (10mL) afforded, after analogous workup a semisolid residue, that waspurified with MPLC on silica. Elution with CHCl₃/MeOH 97:3-20 gave thecrude product (180 mg). Trituration with EtOH yielded 130 mg of the pureproduct 7.

Step 2: Galantamine-3,6-β-D-diglucuronide (8)

2M-NaOH (2.0 mL) was added to a stirred suspension of the aboveglucuronate 7 (130 mg) in MeOH (4 mL), and the mixture left overnight.The solution was then acidified with glacial acetic acid to pH 5.5, thesolvents removed by freeze drying and the product chromatographed onsilica using CHCl₃: MeOH (saturated with dry NH₃) 95:5. gave 48 mg(63.5%) of the product 8.

¹H NMR (CDCl3, 200 MHz): 1.60-1.72 (m, 2H), 1.82-2.6 (m, 10H), 2.88-3.30(m, 3H), 3.50-3.67 (m, 6H), 3.80-4.20 (m, 3H), 4.30-4.70 (m, 1H),4.94-5.30 (m, 6H), 5.76-6.21 (m, 2H), 6.42-6.56 (m, 1H), 6.74-6.86 (m,1H)

Example 10 3-Nicotinoyl-galantamine

A solution of galantamine (431 mg, 1.5 mmol) in dry pyridine (25 mL) wastreated with nicotinoyl chloride (240 mg, 1.7 mmol) and4-N,N-dimethylaminepyridine (5 mg) at 0° and the solution stirred toroom temp. for 2 hrs. followed by heating to 450 for 1 hr. The reactionmixture was poured on water (150 mL) and the pH adjusted to 8.0 followedby extraction with dichloromethane. The organic extract was washed withwater and brine, dried (sodium sulphate) and evaporated to give thecrude product (480 mg, 81.5%)

¹³H NMR (DMSO-d6) δ 27.7, 34.3, 41.7, 47.8, 53.6, 55.9, 60.3, 63.2,86.2, 111.5, 121.3, 122.1, 122.7, 126.0, 129.2, 130.6, 131.9, 136.4,143.9, 146.5, 150.4, 151.5, 166.0.

This product was converted to the dihydrobromide salt by dissolution ina minimum amount of warm 40% hydrobromic acid followed by cooling andobtained as colorless crystals.

Anal. calcd. for C₂₃H₂₄N₂O₄.2HBr.0.33H₂O C, 49.31; H, 4.80; N, 5.00.Found C, 49.10; H, 5.05; N, 4.85.

Example 11 (+−)-8-fluorogalantamine

Step 1: 2-Fluoro-5-hydroxy-4-methoxy benzaldehyde (1)

Sulphuric acid (50 ml, 95-98%) was heated with stirring to the 90-95° C.under a dry nitrogen and 4,5-dimethoxy-2-fluoro benzaldehyde (10.1 g,54.8 mmol) added quickly and this mixture was stirred at the sametemperature for 3.5 h. Reaction was followed by HPLC and found to becomplete after this time. The reaction mixture was poured on crushed ice(150 g) and the white slurry obtained was heated to 65° C. and allowedto cool in the fridge overnight. The white precipitate was filtered andwashed with water (2×100 ml). The wet cake was dried in the desiccatorunder reduced pressure to afford the product (7.6 g, 82%, HPLC 95%,m.p.: 146-148) as off white crystals.

Step 2:4-Fluoro-5-{[2-(4-hydroxyphenyl)ethylamino]-methyl]-2-methoxy-phenol (2)

A solution of 1 (7.6 g, 45 mmol) and tyramine (6.7 g, 49 mmol) in drytoluene (250 ml) and n-butanol (250 ml) was heated and stirred to refluxfor 5 h on the Dean-Stark apparatus to remove the water. Reactiondevelopment was controlled by TLC (MeOH:CH₂Cl₂ 1:9) and reaction wasfound to be complete after this time. Solvents were rotoevaporated andresidue was dissolved in dry methanol (500 ml). NaBH₄ (1.8 g, 45 mmol)was added at the temperature 0-5° C. and this mixture was stirredovernight while the temperature was raised to room temperature and awhite solid precipitated from the reaction mixture. The solid wasfiltered and washed with cold methanol (2×50 ml). The white, wet cakewas dried in the desiccator at reduced pressure to give the product (9.6g, 74%, HPLC >99%) as a white powder. The filtrate was rotaevaporated togive a brown slurry (3.6 g), which was chromatographed on silica(dichloromathane/methanol, gradient 0-10%) to give another (2.5 g, 19%,HPLC >99%) of product as a off white powder (total yield 93%, m.p.:160-162° C.).

¹H NMR (MeOD, 200 MHz): 2.69 (s, broad, 4H), 3.66 (s, 2H), 3.80 (s, 3H),6.66-6.77 (m, 4H), 6.96-7.00 (m, 2H).

Step 3:N-[(2-fluoro-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphenyl)ethyl]-formamide(3)

To a suspension of 2 (7.63 g, 26.1 mmol) in dioxane (50 ml) a solutionof ethyl formiate (3.1 ml, 37.7 mmol), DMF (1.5 ml) and formic acid(0.25 ml, 6.62 mmol) was added dropwise and the reaction mixture washeated under argon to reflux for 10 h. The reaction development wascontrolled by HPLC and showed complete conversion after this time.Volatiles were removed under reduced pressure, the residue was dissolvedin methanol (32 ml) and poured on crushed ice (160 ml), the whiteprecipitate formed was stirred magnetically for 1 h, filtered, washedwith water (3×100 ml) and dried to weight to afford the product (6.8 g,81.3%, HPLC >99%, m.p.: 153-168° C.) as a white powder.

¹H NMR (DMSO, 200 MHz): 2.49-2.67 (m, 2H), 3.15-3.29 (m, 2H), 3.75 (s,3H), 4.28-4.35 (d, 2H, J₂=13.89 Hz), 6.64-6.95 (m, 6H), 7.84 (s, 0.5H),8.20 (s, 0.5H), 8.95-9.00 (d, 1H, 10.17 Hz), 9.18-9.20 (d, 1H, J=2.44Hz).

Step 4:4α,5,9,10,11,12-Hexahydro-1-fluoro-3-methoxy-11-formyl-6H-benzofuro[3a,3,2-ef]benzazepine-6-one(4)

To the vigorously stirred biphasic mixture of potassium carbonate (13.2g, 95.5 mmol) and potassium hexacyanoferrate (28 g, 85.4 mmol) intoluene (580 ml) and water (120 ml), preheated to 50° C., finelypulverized 3 (6.83 g, 21.4 mmol) was added in one portion and thissuspension was heated at 50-60° C. with intense stirring for 1 h. Afterthis time the reaction mixture was filtered trough the pad of celite,the toluene phase separated and the water phase was extracted withtoluene (2×100 ml). The combined organic phases were dried (Na₂SO₄) androto-evaporated under reduced pressure to afford the product (1.3 g,19%, HPLC 98%) as a white powder.

¹H NMR (DMSO, 200 MHz): 1.75-1.93 (m, 1H), 2.15-2.30 (m, 1H), 2.73-2.83(m, 1H), 3.00-3.12 (m, 1H), 3.40 (s, 4H), 3.98-4.13 (m, 1H), 4.28-4.35(m, 0.5H), 4.51-4.97 (m, 2H), 5.27-5.34 (d, 0.5H, J=15.45 Hz), 5.94-6.00(d, 1H, J=10.37 Hz), 6.77-6.86 (m, 1H), 7.15-7.26 (m, 1H), 8.10-8.15 (d,1H, J=8.99 Hz)

¹³C NMR (DMSO, 200 MHz): 34.02, 37.21, 37.32, 45.45, 49.33, 49.53,56.05, 87.29, 100.20, 100.34, 100.77, 100.90, 114.55, 114.93, 115.08,126.66, 130.83, 130.93, 143.12, 143.43, 143.64, 143.76, 144.29, 144.52,162.39, 162.62, 194.77.

Step 5:1-Bromo-4a,5,9,10-tetrahydro-3-methoxy-spiro[6H-benzofuro[3a,3,2-ef][2]benzazepine-6,2′-[1,3]dioxolane]-11(12H)-carboxaldehyde(5)

To the solution of 4 (1.084 g, 3.42 mmol) in toluene (10 ml) a solutionof 4-toluene sulphonic acid (0.02 g, 0.116 mmol) in 1,2-propane-diol(1.13 ml) was added and the mixture heated to the reflux for 1 h whilethe water was removed using a Dean-Stark apparatus. Another portion of4-toluene sulphonic acid (0.05 g) in 1,2-propanediol (0.65 ml) was addedand heating continued for another 5 h. Reaction development wascontrolled by HPLC and the reaction found to be complete after thistime. The reaction mixture was cooled to room temperature and extractedwith acetic acid (2×25 ml, 10% in water), sodium hydrogen carbonate(2×25 ml, 10% in water) and brine (1×25 ml). The toluene solution wasdried (Na₂SO₄) and evaporated to give a crude product (1.32 g) as anamber oil. This was crystallized using i-propanol and ligroin to giveproduct (0.92 g, 72%), as a colourless crystals.

¹H NMR (CDCl₃, 200 MHz): 0.74-2.66 (m, 10H), 2.98-4.86 (m, 8H),5.44-5.74 (m, 1H), 6.34-6.39 (m, 1H), 7.98-8.03 (m, 1H).

(+−)-8-Fluoro-Narwedin (6)

To the solution of 5 (0.91 g, 2.43 mmol) in dry THF (15 ml) lithiumaluminium hydride (1.21 ml, 2.3 mol suspension in THF) was added at 0-5°C. under a continuous stream of dry nitrogen and this mixture wasstirred for 1 h. Another portion of lithium aluminium hydride (0.605 ml,2.3 mmol suspension in THF) was added and stirring continued foradditional 1 h while the temperature raised slowly to room temperature.Reaction development was controlled by HPLC and no starting material wasdetected after this time. The reaction mixture was quenched withwater/THF 1:1 (20 ml) and volatiles removed under reduced pressure. Theresidue was dissolved in 2N-hydrochloric acid (25 ml) and stirred atroom temperature for 30 min. The clear solution was than treated withammonia to pH 12 and extracted with ethyl acetate (3×50 ml). Thecombined organic phases were dried (Na₂SO₄), treated with charcoal,filtered and evaporated to dryness to give 720 mg of the crude productas an brown oil. Chromatography on silica using 7 N NH₃ in MeOH:CH₂Cl₂5:95 as solvents afforded the product (590 mg, yield 80%, HPLC 97%) asan amber oil.

¹H NMR (CDCl₃, 200 MHz): 1.77-1.84 (m, 1H), 2.09-2.24 (m, 1H), 2.38 (s,3H), 2.60-2.71 (m, 1H), 2.98-3.11 (m, 3H), 3.64-3.72 (m, 4H), 4.03-4.11(d, 1H, J=15.65 Hz), 4.65 (s, 1H), 5.93-5.98 (d, 1H, J=10.56 Hz),6.40-6.46 (d, 1H, J=11.34 Hz), 6.84-6.89 (m, 1H)

¹³C NMR (CDCl₃, 200 MHz): 33.41, 37.22, 43.18, 49.42, 49.46, 51.91,51.99, 54.13, 56.20, 88.12, 99.99, 100.58, 114.98, 115.34, 127.31,131.33, 131.43, 142.84, 143.51, 143.71, 144.11, 152.42, 157.18, 194.13.

(+−)-8-fluorogalantamine (7)

To the solution of 6 (500 mg, 1.64 mmol) in dry THF (30 ml) L-Selectride(1.50 ml, 1 M solution in THF) was added dropwise at −5 to 0° C. underdry nitrogen and this mixture was stirred at the same temperature for 30min. The reaction was monitored by HPLC and no starting material wasdetected after this time. The reaction was quenched using water/THF 2:1(50 ml) and solvents were removed under reduced pressure. The residuewas dissolved in 2N-hydrochloric acid (100 ml) and kept overnight in thefridge. The aqueous solution was than washed with diethyl ether (2×30ml) and ammonia was added to pH 12. The aqueous phase was extractedusing ethyl acetate (3×100 ml), the combined organic phases were washedwith brine (50 ml), dried (Na₂SO₄) and evaporated to afford the crudeproduct (515 mg) as a clear, slightly yellow oil which was purified bychromatography on silica using MeOH:CH₂Cl₂ 9:1 to afford the product(0.46 g, 92%, HPLC >99%) as a white powder.

¹H NMR (CDCl₃, 400 MHz): 1.25 (s, 1H), 1.55-1.67 (m, 1H), 1.92-2.10 (m,2H), 2.41 (s, 4H), 2.62-2.70 (m, 1H), 2.98-3.29 (m, 2H), 3.72-3.78 (d,1H), 3.81 (s, 3H), 4.07-4.20 (m, 2H), 4.60 (s, 1H), 6.03 (s, 2H),6.47-6.49 (d, 1H),

¹³C NMR (CDCl₃, 400 MHz): 30.11 (C-5), 34.31 (C-9), 43.10 (N—CH₃), 49.21(C-8a), 52.15 (C-10), 54.32 (OCH₃), 56.55 (C-12), 62.37 (C-6), 89.29(C-4a), 99.86 (C-2), 100.16 (C-12a), 126.89 (C-12b), 134.25 (C-8),134.30 (C-7), 142.09 (C-3a), 144.23 (C-3), 154.31 (C-1), 156.69 (C-1).

(−)-8-fluorogalantamine

The enantiomers of (+−)-8-fluorogalantamine were separated using chiralpreparative column chromatography (Chiracel OD, 5 μm, 5 □ 50 cm, 80%n-heptane/20% i-PrOH) to afford two isomers which were converted to thecorresponding hydrobromide salts. The progress and the result of thischiral separation was analyzed by chiral HPLC (Chiracel I OD-H, 80%n-heptane+0.1% diethyl amine/20% i-PrOH). The crystal structure of (−)2.HBr was determined thus confirming the expectation, that (−)-8-fluoro

ga

lanth

iamine has the same absolute configuration as (−)galantamine.

Example 12 Galantamine, 2-propylpentanoate (ester)

(−)Galantamine (287 mg, 1 mmol), 2-propyl-pentanoic acid (216 mg, 1.5mmol), 4-dimethylaminopyridine (244 mg, 2 mmoles) are added to dryCH₂Cl₂ and stirred for 5 min. A solution of dicyclohexylcarbodiimide(DCC, 2 ml of a 1M solution in CH₂Cl₂) was added in increments and themixture stirred for 20 h under argon. After completion of reaction (asdetermined by TLC, MeOH/CH₂Cl₂ 10:90, visualization with molybdatophosphoric acid) the precipitate was filtered using Hyflo (=diatomaceousearth) and the filtrate was washed with 10% NaHCO₃ and water. Theorganic phase was evaporated and the crude product obtained purified bypreparative chromatography using a gradient of 0 to 8% methanol andmethylene chloride with UV detection. The pure product was isolated byevaporation of the appropriate fractions as a white solid.

¹H NMR (CDCl₃): 0.84 (6H, m); 1.28 (6H, m); 1.57 (3H, m); 2.20 (6H, m);2.52 (1H, m); 3.11 (1H, m); 3.42 (1H, m); 3.79 (4H, m); 4.28 (1H, m);4.56 (1H, m); 5.31 (1H, m); 5.91 (1H, m); 6.32 (1H, m); 6.59 (2H, q).

Following the same procedure the following examples were prepared:

Example No. R ¹H NMR (CDCl₃) 13

1.38 (9H, s); 1.67 (1H, m); 2.11 (2H, m); 2.48(3H, s); 2.59 (1H, m);2.97 (2H, m); 3.29(2H, m); 3.78 (3H, s); 3.65 (2H, m); 4.10 (1H, m);4.44 (1H, m); 5.31 (1H, m); 5.81 (1H, m); 6.34 (1H, m); 6.61 (2H, m);7.19 (5H, m) 14

1.31 (9H, s); 1.54 (1H, m); 1.96(2H, m); 2.32(3H, s); 2.56 (1H, m); 3.01(2H, m); 3.30(2H, m); 3.78 (3H, s); 3.65 (2H, m); 4.08 (1H, m); 4.42(1H, m); 5.23 (1H, m); 5.38 (2H, m); 5.79 (1H, m); 6.28 (1H, m); 6.51(2H, m); 7.13 (8H, m) 15

1.41 (2H, m); 1.61 (4H, m); 1.81 (1H, m); 2.03 (2H, m); 2.38 (6H, m);2.69 (1H, m); 3.09 (3H, m); 3.30 (1H, m); 3.70 (1H, m); 3.83 (3H, s);4.08 (1H, m); 4.55 (1H, m); 5.29 (1H, m); 5.90 (1H, q); 6.31 (1H, d),6.63 (2H, m) 16

1.49 (9H, m): 1.54 (9H, m); 2.13 (2H, m); 2.49 (4H, m); 2.63 (1H, m);3.17 (4H, m); 3.78 (5H, m); 4.57 (2H, m); 5.33 (1H, m); 6.07 (1H, m);6.31 (1H, m); 6.63 (2H, q); 7.27 (2H, m) 17

1.03 (3H, t); 1.54 (1H, m); 2.00 (2H, m); 2.25 (2H, m); 2.55 (3H, s);2.64 (1H, m); 2.94 (1H, m); 3.02 (1H, m); 3.54 (1H, m); 3.76 (3H, s);4.09 (1H, m); 4.48 (1H, m); 5.26 (1H, m); 5.76 (1H, m); 6.19 (1H, m);6.56 (2H, q) 18

1.06 (6H, m); 1.41 (1H, m); 1.59 (1H, m); 2.05 (2H, m); 2.32 (3H, m);2.53 (1H, m); 3.01 (1H, m); 3.23 (1H, m); 3.55 (1H, m); 3.78 (3H, s);4.09 (1H, m); 4.48 (1H, m); 5.22 (1H, m); 5.83 (1H, m); 6.47 (1H, d);6.60 (2H, q) 19

1.07 (9H, m); 1.51 (1H, m); 2.00 (2H, m); 2.26 (2H, m); 2.59 (3H, s);2.54 (1H, m); 2.98 (1H, m); 3.02 (1H, m); 3.54 (1H, m); 3.76 (3H, s);4.09 (1H, m); 4.48 (1H, m); 5.26 (1H, m); 5.81 (1H, m); 6.21 (1H, m);6.62 (2H, q)

Example 20 L-Phenylalanine, N-[(1,1-dimethylethoxy)carbonyl]-,(4aS,6S,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester

To solution of (−) galantamine (287 mg, 1.0 mmol) in dry CH₂Cl₂ (30 mL)N-Boc-phenylalanine (400 mg, 1.5 mmol) and triphenyl phosphine (340 mg,1.3 mmol) are added with magnetic stirring followed by the drop-wiseaddition of diisopropyl azodicarboxylate (DIAD) (270 mg, 1.34 mmoles.)to the reaction mixture at −10° C. The reaction was stirred overnight atroom temperature under argon. After the completion of the reaction(TLC-MeOH/CH₂Cl₂ (10:90)) the reaction mixture was filtered and thefiltrate was washed with 10% NaHCO₃ and water. The organic phase wasevaporated and the crude product obtained purified by preparativechromatography using a gradient of 0 to 8% methanol and methylenechloride with UV detection. From the fractions containing the pureproducts these were isolated by evaporation of the solvents. Thisprocedure results in the inversion of configuration on oxygen inposition 6.

¹H NMR (CDCl₃) 1.35 (9H, s); 1.60 (1H, m); 2.05 (2H, m); 2.36 (3H, s);2.59 (1H, m); 2.90 (2H, m); 3.30 (2H, m); 3.58 (3H, s); 3.65 (2H, m);4.08 (1H, m); 4.42 (1H, m); 5.23 (1H, m); 5.79 (1H, m); 6.28 (1H, m);6.54 (2H, m); 7.13 (5H, m)

Example 21 L-Phenylalanine,(4aS,6S,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester

L-Phenylalanine,(4aS,6S,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester was prepared from the compound obtained in example 20 byBoc-deprotection using trifluoro acetic acid in methylene chloridefollowed by the usual workup and resulted in the product as a whitepowder.

¹H NMR (CDCl3) 1.82 (2H, m); 2.05 (2H, m); 2.36 (3H, s); 2.59 (1H, m);2.90 (2H, m); 3.30 (2H, m); 3.58 (3H, s); 3.65 (2H, m); 4.08 (1H, m);4.42 (1H, m); 5.23 (1H, m); 5.79 (1H, m); 6.28 (1H, m); 6.54 (2H, m);7.13 (5H, m)

Example 22L-tyrosine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester

L-tyrosine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester was prepared from the compound of example 13 using the samedeprotection method as in example 21.

¹H NMR (CDCl₃) 1.68 (2H, m); 1.96 (2H, m); 2.32 (3H, s); 2.56 (1H, m);3.01 (2H, m); 3.30 (2H, m); 3.78 (3H, s); 3.65 (2H, m); 4.08 (1H, m);4.42 (1H, m); 5.23 (1H, m); 5.79 (2H, m); 6.28 (1H, m); 6.51 (2H, m);7.13 (4H, dd)

Example 23L-histidine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester hydrochloride

L-histidine-(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ylester hydrochloride was prepared from the compound of example 14 usingHCl in ethyl acetate for deprotection and resulted in the isolation ofthe product as the hydrochloride.

¹H NMR (CDCl3) 2.34 (2H, m); 2.54 (4H, m); 2.78 (1H, m); 3.21 (4H, m);3.79 (5H, m); 4.58 (2H, m); 5.41 (1H, m); 6.18 (1H, m); 6.48 (1H, m);6.65 (2H, q); 7.38 (2H, m)

Example 24 (4aS,6R,8aS)-6H-Benzofuro[3a,3,2-ef][2]benzazepin-6-ol,4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-, hydrogen sulfate (ester)

Chlorsulfonic acid (0.16 g, 1.39 mmol) was added to dry pyridine (1 ml)preheated to 70-80° C. and stirred at the same temperature for 30 min. Asolution of galantamine (0.20 g, 0.70 mmol) in dry pyridine (1 ml) wasadded drop wise and the mixture was stirred overnight at roomtemperature with the formation of a precipitate. MeOH/H₂O 1:1 (5 ml) wasadded and the resulting clear solution was stirred for further 30 min.Volatiles were rotoevaporated and another portion of MeOH (5 ml) wasadded. The resulting fine precipitate was filtered to give (0.21 g,yield 82%, HPLC >99%) of product as a white powder.

IR: 1700.59, 1652.92, 1623.93, 1617.01, 1510.15, 1475.31, 1443.53,1299.82, 1282.40, 1266.98, 1242.70, 1217.83, 1197.48, 1155.3, 1092.40,1070.97, 1053.15, 1023.70, 1007.21, 984.45.

Example 25

General Procedure 1

To the solution of (−)-galantamine hydrobromide (1.0 mole) and triethylamine (4.0 mol) in DCM (30 mL), DMAP (0.5 mol) was added followed byrespective acid chloride or acid anhydride (1.2 mol). The mixture wasstirred overnight at room temperature under argon. The reaction mixturewas washed with 10% NaHCO₃ and brine, dried (Na₂SO₄) and concentrated.The crude compound obtained was purified by column chromatography orrecrystallization to give the pure product.

Using this procedure the following compounds were obtained:

O-Benzoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,benzoate (ester)); yield: 78%

O-3,4-Dichlorobenzoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,3,4-dichlorobenzoate (ester)); off-white solid; mp. 69-70° C.

¹H NMR (200 MHz, CDCl₃) δ (ppm) 8.02 (d, J=1.88 Hz, 1H), 7.81 (dd,J=1.88 Hz, J=8.38 Hz, 1H), 7.38 (d, J=8.32 Hz, 1H), 6.62 (d, J=8.18 Hz,1H), 6.52 (d, J=8.18 Hz, 1H), 6.32 (d, J=10.34 Hz, 1H), 5.89-5.97 (m,1H), 5.51 (t, J=4.43 Hz, 1H), 4.58 (s, 1H), 4.07 (d, J=15.16 Hz, 1H),3.18 (s, 3H), 3.61 (d, J=15.16 Hz, 1H), 3.21-3.45 (m, 1H), 2.96-3.05 (m,1H), 2.66-2.76 (m, 1H), 2.34 (s, 3H), 2.0-2.19 (m, 2H), 1.51-1.59 (m,1H).

O-4-Methoxybenzoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,4-methoxybenzoate (ester)); off-white solid; mp. 183-184° C.

¹H NMR (200 MHz, CDCl₃) δ (ppm) 8.01 (d, J=9.0 Hz, 2H), 8.56 (d, J=8.86Hz, 2H), 6.69 (d, J=8.18 Hz, 1H), 6.58 (d, J=8.2 Hz, 1H), 6.35 (d,J=10.2 Hz, 1H), 6.0-6.07 (m, 1H), 5.56 (t, J=4.49 Hz, 1H), 4.66 (s, 1H),4.15 (d, J=15.18 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 3.68 (d, J=15.18Hz, 1H), 3.29-3.53 (m, 1H), 3.04-3.12 (m, 1H), 2.73-2.81 (m, 1H), 2.41(s, 3H), 2.08-2.26 (m, 2H), 1.58-1.66 (m, 1H).

O-4-Methylbenzoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,4-methylbenzoate (ester)); off-white solid; mp. 71-72° C.

¹H NMR (200 MHz, CDCl₃) δ (ppm) 7.94 (d, J=8.18 Hz, 2H), 7.17 (d, J=8.06Hz, 2H), 6.69 (d, J=8.18 Hz, 1H), 6.58 (d, J=8.2 Hz, 1H), 6.35 (d,J=9.52 Hz, 1H), 6.0-6.08 (m, 1H), 5.57 (t, J=4.43 Hz, 1H), 4.66 (s, 1H),4.17 (d, J=15.18 Hz, 1H), 3.89 (s, 3H), 3.70 (d, J=15.18 Hz, 1H),3.31-3.43 (m, 1H), 3.06-3.13 (m, 1H), 2.74-2.83 (m, 1H), 2.42 (s, 3H),2.38 (s, 3H), 2.08-2.26 (m, 2H), 1.59-1.67 (m, 1H).

O-4-Chlorobenzoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,4-chlorobenzoate (ester)); off-white solid; mp. 72-74° C.

¹H NMR (200 MHz, CDCl₃) δ (ppm) 7.91 (d, J=8.74 Hz, 2H), 7.27 (d, J=8.72Hz, 2H), 6.62 (d, J=8.2 Hz, 1H), 6.52 (d, J=8.2 Hz, 1H), 6.30 (d,J=10.34 Hz, 1H), 5.92-6.0 (m, 1H), 5.5 (t, J=4.36 Hz, 1H), 4.59 (s, 1H),4.09 (d, J=15.18 Hz, 1H), 3.82 (s, 3H), 3.63 (d, J=15.18 Hz, 1H),3.23-3.46 (m, 1H), 2.99-3.06 (m, 1H), 2.66-2.76 (m, 1H), 2.35 (s, 3H),2.0-2.2 (m, 2H), 1.52-1.6 (m, 1H).

O-2-Thenoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,thiophene-2-carboxylate (ester)); off-white solid; mp. 115-116° C.

¹H NMR (200 MHz, CDCl₃) δ (ppm) 7.78 (dd, J=1.2 Hz, J=3.8 Hz, 1H), 7.51(dd, J=1.34 Hz, J=4.96 Hz, 1H), 7.04 (dd, J=3.76 Hz, J=4.98 Hz, 1H),6.69 (d, J=8.18 Hz, 1H), 6.59 (d, J=8.04 Hz, 1H), 6.35 (d, J=10.2 Hz,1H), 6.02 (dd, J=4.7 Hz, J=10.2 Hz, 1H), 5.54 (t, J=4.49 Hz, 1H), 4.63(s, 1H), 4.18 (d, J=15.02 Hz, 1H), 3.87 (s, 3H), 3.71 (d, J=15.18 Hz,1H), 3.31-3.5 (m, 1H), 3.07-3.14 (m, 1H), 2.73-2.83 (m, 1H), 2.42 (s,3H), 2.04-2.26 (m, 2H), 1.6-1.68 (m, 1H).

O-5-Chloro-2-thenoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,5-chlorothiophene-2-carboxylate (ester)); off-white solid; mp. 58-59° C.

¹H NMR (200 MHz, CDCl₃) δ (ppm) 7.5 (d, J=4.04 Hz, 1H), 7.80 (d, J=4.02Hz, 1H), 6.62 (d, J=8.04 Hz, 1H), 6.52 (d, J=8.06 Hz, 1H), 6.31 (d,J=10.2 Hz, 1H), 5.92 (dd, J=4.57 Hz, J=10.2 Hz, 1H), 5.45 (t, J=4.36 Hz,1H), 4.56 (s, 1H), 4.08 (d, J=15.16 Hz, 1H), 3.81 (s, 3H), 3.61 (d,J=15.18 Hz, 1H), 3.21-3.34 (m, 1H), 2.97-3.04 (m, 1H), 2.64-2.74 (m,1H), 2.34 (s, 3H), 1.97-2.19 (m, 2H), 1.5-1.57 (m, 1H).

Example 26

General Procedure 2

To the solution of the corresponding acid acid (13.87 g, 135.8 mmol) inDCM (250 mL) was added DCC (33.62 g, 162.9 mmol) followed by DMAP (3.32g, 27.15 mmol), reaction mixture was stirred for additional 30 minutesat room temperature. To this (−)-galantamine hydrobromide (10.0 g, 27.15mmol) and triethyl amine (4.6 mL, 32.59 mmol) was added, the mixture wasstirred overnight at room temperature under argon. The precipitated DCHUwas removed by filtration and the filtrate was evaporated. Theadditional DCHU was removed by subsequent trituration with cold ethylacetate and filtration. The ethyl acetate solution was roto-evaporatedand the crude product obtained was purified by column chromatography togive the desired product.

2-Methyl-butanoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,2-methyl-butanoate (ester)) was obtained in 53% yield as a solid usingthe general procedure 2.

The same product, identical in every respect (HPLC, m.p., 1H-NMR), wasalso obtained in 58% yield using the general procedure 1.

2-Methyl-propanoyl-galantamine(=(4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]benzazepin-6-ol,2-methyl-propanoate (ester)) was obtained in 63% yield as a solid usingthe general procedure 1.

2-Methyl-propanoyl-galantamine hydrochloride salt

To the solution of 2-methyl-propanoyl-galantamine (150 mg, 0.43 mmol) inethyl acetate (5 mL), ethyl acetate saturated with HCl (5 mL) was addedslowly with stirring at 0° C. The reaction mixture was stirred at roomtemperature for 1 h. Solvent was evaporated and the residue obtained waswashed with dry ether and was dried under high vacuum to give 164 mg(97%) of desired product as an off-white solid.

Anal. calcd for C₂₁H₂₇NO₄ (1.5 HCl): C, 61.2; H, 6.97; N, 3.40. Found:C, 61.62; H, 6.95; N, 3.91.

2-Methyl-propanoyl-galantamine citric acid salt

To the solution of 2-methyl-propanoyl-galantamine (150 mg, 0.43 mmol) inmethanol (5 mL), a solution of citric acid in methanol (5 mL) was addedslowly with stirring at room temperature. The reaction mixture wasstirred at room temperature for 1 h. Solvent was evaporated and theresidue obtained was precipitated from methanol-diethyl ether and wasdried under high vacuum to give 187 mg (81%) of desired product as aoff-white solid.

Anal. calcd for C₂₇H₃₅NO₁₁ (1.0H₂O): C, 57.14; H, 6.57; N, 2.47. Found:C, 57.43; H, 6.48; N, 2.53.

Example 27

General Procedure 3

To a stirred solution of (−)-galantamine hydrobromide (1.10 mmol) inpyridine (6 mL) at 0° C. under nitrogen, the corresponding acid chloride(2.2 mmol) was added and the mixture was stirred until a TLC showed thereaction to be complete. Then CH₂CL₂ (10 mL) and water (10 mL) wereadded and stirring was continued for 30 min. The organic layer wasseparated, washed with water (2×10 mL), dried over anhydrous MgSO₄ andthe solvent removed. The residue was purified by flash chromatographygiving the product identical in all respects to O-Benzoyl-galantamine.

Example 28 Synthesis of R1-pyridinoyl-galantamine

In addition to the examples provided above, the following compounds wereprepared by the described general procedures:

TABLE 6 MF MW Substituent R1 C₂₁H₂₅NO₄ 355.43 cyclopropanecarboxylateC₂₄H₂₃Cl₂NO₄ 460.3 3,4-dichlorobenzoate C₂₈H₃₃NO₄ 447.574-tert-butylbenzoate C₂₅H₂₃ClF₃NO₄ 493.914-chloro-3-(trifluoromethyl)benzoate C₂₅H₂₃F₃N₂O₆ 504.464-nitro-3-(trifluoromethyl)benzoate C₂₅H₂₄F₃NO₄ 459.474-(trifluoromethyl)benzoate C₂₄H₂₃Cl₂NO₄ 460.36 2,4-dichlorobenzoateC₂₄H₂₄N₂O₆ 436.47 4-nitrobenzoate C₂₄H₂₄ClNO₄ 425.92 3-chlorobenzoateC₂₅H₂₄F₃NO₄ 459.47 3-(trifluoromethyl)benzoate C₂₄H₂₄N₂O₆ 436.473-nitrobenzoate C₂₄H₂₃Cl₂NO₄ 460.36 3,5-dichlorobenzoate C₂₆H₃₀N₂O₄434.54 3-(dimethylamino)benzoate C₂₅H₂₇NO₄ 405.50 3-methylbenzoateC₂₄H₂₄ClNO₄ 425.92 2-chlorobenzoate C₂₄H₂₃F₂NO₄ 427.452,4-difluorobenzoate C₂₄H₂₃Cl₂NO₄ 460.36 2,5-dichlorobenzoate C₂₄H₂₄FNO₄409.46 4-fluorobenzoate C₂₆H₃₀N₂O₄ 434.54 4-(dimethylamino)benzoateC₂₄H₂₆N₂O₄ 406.49 4-aminobenzoate C₂₇H₃₂N₂O₄ 448.574-(dimethylamino)-3-methylbenzoate C₂₅H₂₅NO₆ 435.482H-1,3-benzodioxole-5-carboxylate C₂₆H₂₇NO₅ 433.51 4-acetylbenzoateC₂₂H₂₃NO₄S 397.50 thiophene-3-carboxylate C₂₁H₂₃N₃O₄ 381.441H-imidazole-5-carboxylate C₂₁H₂₂N₂O₅ 382.42 1,3-oxazole-5-carboxylateC₂₁H₂₂N₂O₄S 398.48 1,3-thiazole-5-carboxylate C₂₁H₂₂N₂O₄S 398.481,3-thiazole-2-carboxylate C₂₆H₂₇NO₆ 449.51 2-(acetyloxy)benzoate

Example 29

Preparation of Mouse Brain Homogenate

The brain is removed from the animal (mouse), shock frozen in liquidnitrogen and stored at minus 80° C. until use. Before preparing theextract, the frozen brain is thawed on ice and the weight is determined.Ice cold buffer (130 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 5 mMGlucose, 5 mM HEPES, pH 7.4) is added to the thawed mouse brain (1:4,weight to volume, resulting in a 20% brain homogenate). The tissue isthen homogenized in a potter homogeniser on ice, moving the piston upand down 11 times at 240 rpm. The freshly prepared mouse brainhomogenate is divided into aliquots.

Example 30

Procedure for Measuring the Inhibition of Brain Esterase. Results Shownas FIG. 1

A modified Ellmann's esterase test is used. Briefly the method relies onthe cleavage of the substrate acetylthiocholine to acetate andthiocholine. The latter reacts with DTNB(5,5′-Dithiobis-(2-nitro-benzoicacid) to a yellow compound, which can bequantified spectrometrically. The incubation buffer contains 51 mmol/lsodium phosphate buffer and 0.05% Tween 20, at pH 7.2 and issupplemented with 100 mg/l DTNB, and 0.2% mouse brain homogenate(prepared as described in Example 29). The compound to be investigatedis added to the desired concentration. The mixture is brought to 37° C.and the reaction is started by addition of 200 μM acetylthiocholine.A₄₀₅ is measured in is intervals in a microplate reader for 40 s. Thelinear parts of the absorption-time-curves represent the starting speedof the enzymatic reaction and are used for the calculation of thereaction speed. The slope of the curve corresponds to the reactionspeed. The inhibition is expressed as percent of the non inhibitedreaction according to following equation:% Inhibition=100*(1−(Slope_(inhibited)/Slope_(noninhibited))).

Example 31

Procedure for Determination of Prodrug Cleavage in Mouse BrainHomogenate.

To aliquots of mouse brain homogenate as prepared according to Example13 pro-galantamine derivatives are added and adjusted to a finalconcentration of 10 μM of the prodrug. At the end of the incubationtime, 12 μl 0.1 M NaOH and 100 μl saturated KCl are added to the 0.1 mlreaction mixture and mixed thoroughly. The remaining prodrug and thereleased galantamine are extracted by 200 μl toluene. The tolueneextraction step is repeated twice using 150 μl toluene and the obtainedextracts are pooled, dried, dissolved in 50% Methanol and used forsubsequent HPLC analysis.

Example 32

Investigation of the allosteric modulation effect of drug candidates onnicotinic acetylcholine receptors (nAChRs) expressed in HEK-293 cells byelectrophysiology. Results shown as FIG. 4.

Single HEK-293 cells expressing either human α4β2, human α3β4, orchimeric chicken α7 (with mouse 5HT₃) nAChR were plated onfibronectin-coated cover slips for 3 days before measurement. Theselected nAChR containing cells were placed in the recording bath,filled with extracellular buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2mM CaCl₂, 10 mM D-glucose, 10 mM HEPES, pH 7.3, approximately 300 mOsm).Patch-clamp system consisted of an inverted microscope (Zeiss, Germany),computer-controlled patch-clamp amplifier with PatchMaster software(HEKA, Germany), tubing perfusion system (ALA, USA) together with aU-tube applicator (IMM, Germany) and dual micromanipulators. The patchpipettes were pulled from fire-polished, 100 mm long and 1.5 mm width,single borosilicate glass capillaries (WPI, Germany). A programmablepuller (Sutter, USA) was used to prepare a twin pair of ready for usepipettes. Each patch pipette (resistance 4-8 MΩ) was used only once.Pipettes were filled with an internal buffer (140 mM CsCl, 11 mM EGTA,10 mM HEPES, 2 mM MgCl₂, pH 7.3, approximately 300 mOsm) and connectedto the working electrode. Working and reference electrodes forexperiments were made from daily renewed, freshly chlorinated silverwire (40 mm×0.4 mm) and were connected to a headstage circuit of thepatch-clamp amplifier. Patching was done using rectangular test pulseswith an amplitude of −1 mV and a duration of 20 ms. Immediately afterformation of the gigaseal the holding potential of −70 mV was applied tothe patch electrode and whole-cell recordings were established by usingnegative pressure pulses. All necessary compensations for fast and slowmembrane capacitance and serial resistance transients were automaticallyset within the PatchMaster software. Whole-cell currents were evoked bythe application of nicotine at the EC₅₀ for each appropriate nAChRsubtype (α4β2 and α3β4 EC₅₀=30 μM, chimeric α7 EC₅₀=3 μM). To evaluatean allosteric potentiating ligand (APL) effect of selected compounds oneach subtype of nAChR, they were added to stimulating nicotine solutionsat the following concentrations: 1, 5, 10, 50, 100, 500, 1,000, 5,000and 10,000 nM, and solutions were applied to the cell surface during 500ms pulses through the U-tube, and then corresponding currents, digitizedto 10 kHz, were recorded for 10 s. Consecutive current stimulations weredone with a 2 min interval to avoid nAChR desensibilisation and toensure full exchange of stimulating solutions. The averaged peakamplitudes of the currents, measured in the presence of selectedcompound concentrations, were compared with those determined in theabsence of compounds (control) and they were calculated as % of control.The measurements of an APL effect of particular compound were repeatedon a minimum of five cells to obtain the mean values+/−SD. Mean valuesof the observed APL effect, which did not exceed 15% were treated asinsignificant. To present a concentration-dependent APL effect ofparticular compound, the corresponding % of control values+/−SD wereplotted against the concentrations used.

Example 33

Pharmacokinetics of Pro-Galantamine Gln-1062 (3 mg/kg) in the Mouse.(Results Shown as FIG. 5.)

Study Objective

Determination of the pharmacokinetic profiles of Gln-1062 and itscleavage product galantamine in blood and brain. Determine thebrain-to-blood concentration ratios of Gln-1062 and its cleavage productgalantamine, and assess the blood-brain barrier penetration capacities.

Study Plan

Bioanalysis

Analytical method for estimation of Gln-1062 was evaluated for itslinearity, precision & accuracy and recovery in SAM blood and brainhomogenate using LC/MS/MS.

LC/MS/MS Parameters

The parameters of chromatographic conditions and extraction conditionsfor the Gln-1062 and Galantamine analysis were

Chromatographic Parameters:

-   -   Column: Phenomenex Synergi, Polar-RP 80 A, C18, 75×2.0 mm, 4μ

Mobile Phase

-   -   Mobile Phase Buffer: 40 mM Ammonium Formate, pH 3.5    -   Aqueous Reservoir (A): 10% Buffer, 90% Water    -   Organic Reservoir (B): 10% Buffer, 90% Acetonitrile    -   Flow rate: 0.450 mL/min

Gradient Programme:

Gradient Time (min) Curve % A % B 0 1 100 0 1.2 1 60 40 3 1 0 100 3.1 1100 0 5 1 100 0

Divert Valve Time Schedule:

Divert Valve Time (min) Waste MS 0 x 1.2 x 4.5 x

-   -   Run time: 5.0 min    -   Column oven temperature: Ambient    -   Auto sampler temperature: Ambient    -   Auto sampler Wash: Water:acetonitrile:isopropanol with 0.2%        formic acid, 1:1:1(v/v/v)

Retention time: Gln-1062: 3.33 ± 0.05 min. Galantamine: 2.44 ± 0.05 minMetoprolol: 2.80 ± 0.05 min.

Mass Parameters (API 3200):

-   -   Mode: MRM    -   Polarity: Positive    -   Ion source: Turbo spray    -   Analyte: Gln-1062 (Q1 Mass 392.4; Q3 Mass 213.2)        -   Galantamine (Q1 Mass 288.3; Q3 Mass 213.1)    -   ISTD: Metoprolol (Q1 Mass 268.4; Q3 Mass 116.2)

Source/Gas Parameters:

Curtain gas (CUR) 10 Collision gas  5 (Collision associatedDissociation) CAD Ion Spray Voltage (IS) 5500 V Temperature (TEM) 575°C. GS1 55 GS2 45 Ihe ON

Compound Parameters:

Parameter Galantamine Metoprolol Gln-1062 Declustering Potential (V) 4535 50 Entrance Potential (V) 10 10 10 Collision Cell Entrance Potential20 20 20.00 (V) Collision Energy (eV) 32 26 32 Collision Cell ExitPotential (V) 4.5 2 5 Dwell Time (milliSec) 200 200 200Extraction Procedure:

The Extraction procedure, including preparation of STD, QC and studysamples, and preparation of calibration curve standards for recovery isshown in FIGS. 8, (A) and (B).

Method Evaluation

This method was evaluated for linearity, precision & accuracy andrecovery of Gln-1062 in SAM blood and brain homogenate.

A. Linearity, Precision & Accuracy

-   -   A single standard curve and six replicates each of three quality        control (QC) levels (18 total QCs) were extracted and analyzed.        The linearity of the calibration curve was determined by a        weighed least square regression analysis.

Acceptance Criteria

-   -   i. At least six out of nine standards must have an accuracy of        ±15% from nominal, except at the lower limit of quantitation        (LLOQ) where ±20% is acceptable.    -   ii. Two-thirds of the batch QCs and at least half of the QCs at        each level must have a calculated accuracy of ±15% from nominal.    -   iii. Intra-assay Mean Precision and Accuracy        -   1. Four out of six QCs must be available to determine            accuracy and precision.        -   2. The intra-assay coefficient of variation (% CV) of each            QC level must not exceed 15% and the accuracy of the mean            value for each validation to be accepted.    -   Gln-1062 linearity, precision and accuracy in blood and brain        homogenate matrices were evaluated.

B. Recovery

The recovery of Gln-1062 from the blood and brain homogenate matriceswere also evaluated.

Recovery was determined by quantifying the concentration of the analytesin extracted matrix QC samples with a standard curve prepared inpost-extract (blank extracts) sample matrix as described above insection B of the extraction procedure, entitled “Preparation ofCalibration Curve Standards for Recovery.”

Gln-1062 recovery in blood and brain homogenate matrices were evaluated.

Animal Study

Study Design

No. of Dose Dose Animals Sample time Dose Conc. Volume Dose for eachpoints Animal Test item (mg/kg) (mg/mL) (mL/kg) route time point (hr)Male SAM Gln-1062 3 0.2 15 i.v, bolus 3 Predose, 0.05, 25-33 gm (tailvein) 0.10, 0.17, 0.33, 0.50, 0.83, 1.33, 2.0 and 4.0Sample CollectionCollection of Blood:

Blood samples were collected from the retro-orbital plexus. 0.5 ml ofblood was collected into a pre-labeled polypropylene micro centrifugetube containing sodium citrate as the anticoagulant, and kept on ice.

Blood was mixed gently with anticoagulant and an aliquot of 50 μL ofblood sample immediately precipitated as described above in section A ofthe extraction procedure, entitled “Preparation of STD, QC and StudySamples.” Remaining volume of blood sample at each time point frozen ondry ice.

All the blood samples were transferred to analytical department andfrozen at −80±10° C. until analysis.

Collection of Brain:

Immediately after blood withdrawal, brain was perfused with phosphatebuffer saline (pH 7.4), removed and frozen on dry ice.

All the brain samples were transferred to analytical department andfrozen at −80° C. until analysis.

Brain Homogenate Preparation:

Brain samples were thawed on ice and weighed. n appropriate volume icecold homogenizing media (methanol:water::20:80,v/v) added. n ice,homogenized the brain sample with the polytron homogenizer and make upthe volume with homogenizing media to get 1 gm of brain per 4 mL ofhomogenate. After homogenizing, immediately freezed the brain homogenatesamples at −80° C. until analysis.

Example 34

Behavioural index for gastro-intestinal side effects in ferretsfollowing application of galantamine and several R1-pro-galantamines,respectively. Results shown in FIG. 6.

Test System

Fourteen adult male Putoris furo ferrets (Marshall BioResources (NorthRose, USA)), weighting 750-1000 grams on the day of experimentation wereused in the present study. In agreement with the sponsor four out of thefourteen animals were included in two experimental groups, ferrets #1,2, 3 and 4).

Animal Housing

The acclimatization of the animals lasted at least 5 days. At receipt,animals were collectively housed in cages at Syncrosome's premises. Theyhad free access to food and drinking water ad libitum.

Test Item and Reference Compound

During this study, one reference compound (Galantamine) and one Galantoscandidate compound (GLN979) were tested at two doses each. Bothcompounds were administered I.P. at doses and concentrations.

Details of the different compound shipments are presented in Table 7.

TABLE 7 Receipt Compound Nov. 11^(th), 2007 Nov., the 21^(st), 2007Nov., the 29^(th), 2007 Galantamine 48.0 mg 256.0 mg (One vial) — GLN97948.0 mg — 262.6 mg 2-Hydroxy U.I: (One vial) 7.3 g + 7.3 g (Two ≈4.7 g +≈5.0 g propyl-β- vials) cyclodextrin NaCl U.I: (One vial) 3.0 g (Onevial) —

As requested by the sponsor, the same vehicle (15%2-Hydroxypropyl-β-cyclodextrin/96 mM NaCl) was used for both Galantamineand GLN979 preparation. A detailed solubilization protocol was sent bymail by Galantos to Syncrosome and received on Nov. 5, 2007.

Test Compound (GLN979)

Nature GLN979. Molar mass U.I. Administration dose 20 and 40 mg/kg B.W.Administration route I.P. Vehicle 15% 2-Hydroxypropyl-β-cyclodextrin/96mM NaCl.Reference Compound (Galantamine)

Nature Galantamine Molar mass U.I. Administration dose 3 and 20 mg/kgB.W. Administration route I.P. Vehicle 15%2-Hydroxypropyl-β-cyclodextrin/96 mM NaCl.I.P. Administrations

For the 4 experimental groups, the administrations were performed inunanaesthetized animals through the I.P. route at T₀.

Emesis Test

After I.P. administration of the compound solution, the animals werecontinuously observed by a trained technician for four hours. Duringthat period, the number of episodes of vomiting (series of retchesleading to the expulsion of part of the gastro-intestinal content) wererecorded.

Behavioural Observation

During the 4 hours-observation period, several side-effects (salivation,shivering, respiratory problems and diarrhea) were also observed. Foreach of these behaviours, a scoring method was determined with thesponsor. Depending on its severity, each parameter were quantified as:

-   -   “None” (None): Behaviour not observed.    -   “Moderate” (Mod.): Behaviour observed with a low frequency        and/or a low intensity.    -   “Intense” (Int.): Behaviour observed frequently and/or        continuously and/or at a high intensity.        Inclusion Criteria

All the animals receiving I.P. administration of the reference or thetest compound were included in the study regardless of the pattern ofboth their emetic responses and behavious.

Example 35

Reversal from scopolamine-induced amnesia in mice, results shown as FIG.6

Drug Preparation

Gln 1062, Gln 0979 and galantamine were dissolved in15%—hydroxypropyl-β-cyclodextrin in 96 mM NaCl (isotonic) supplied bythe sponsor. Gln 1062 and Gln 0979 were used in concentrations of 0.01,0.03, 0.1 and 0.2 mg/ml, which when given in a volume of 10 ml/kg resultin doses of 0.1, 0.3, 1 and 2 mg/kg i.p., respectively. Galantamine wasused in concentrations of 0.03, 0.1, 0.2 and 0.5 mg/ml, which when givenin a volume of 10 ml/kg result in doses of 0.3, 1, 2 and 5 mg/kg i.p.,respectively.

Control animals received 15%—hydroxypropyl-β-cyclodextrin in 96 mM NaClinjection as a vehicle.

Nicotine ((−)-Nicotine hydrogen tartrate salt, Sigma, France),scopolamine (−(−)scopolamine hydrochloride, Sigma, France) weredissolved in saline (0.9% NaCl, Aguettant, France) at the concentrationof 0.04 and 0.1 mg/ml, respectively. They were administrated at a dosagevolume of 10 ml/kg to achieve doses of 0.4 and 1 mg/kg, respectively.

Test Animals

Four to five week old male CD-1 mice (Janvier; Le Genest St Isle—France)were used for the study.

They were group-housed (10 mice per cage) and maintained in a room withcontrolled temperature (21-22° C.) and a reversed light-dark cycle (12h/12 h; lights on: 17:30-05:30; lights off: 05:30-17:30) with food andwater available ad libitum.

Experimental Design

The potential cognitive enhancing property of Gln 1062, Gln 0979 andgalantamine were evaluated in scopolamine-treated mice in the T-mazealternation model under the same experimental conditions. Both Gln 1062and Gln 0979 were tested in doses of 0.1, 0.3, 1 and 2 mg/kg i.p.Galantamine was tested in doses 0.1, 0.3, 1, 2 and 5 mg/kg i.p. Nicotinewas tested in a dose of 0.4 mg/kg i.p. All these compounds wereadministrated immediately after the injection of 1 mg/kg i.p.scopolamine (20 min prior to the T-maze trial) used to induce memorydeficit.

Memory performance was assessed by the percentage of spontaneousalternation in the T-maze. The number of alternation in saline-injectedmice was used as the base level of unaltered memory performance.

Mice were housed 10 per cage. Each mouse in a cage was randomly assignedby a unique number (1 to 10) written on the tail with permanent ink.

Gln 1062, Gln 0979 and galantamine were tested separately in three setof experiments with different animals. Each set of experiments wasdivided in series of daily experiments that always comprises at leastone representative of each of Saline/vehicle, Scopolamine/vehicle andScopolamine/Nicotine (0.4 mg/kg) groups.

Measurement

The T-maze apparatus was made of gray Plexiglas with a main stem (55 cmlong×10 cm wide×20 cm high) and two arms (30 cm long×10 cm wide×20 cmhigh) positioned at 90 degree angle relative to the left and right ofthe main stem. A start box (15 cm long×10 cm wide) was separated fromthe main stem by a guillotine door. Horizontal doors were present toclose off specific arms during the force choice alternation task.

The experimental protocol consists of one single session, which startswith 1 “forced-choice” trial, followed by 14 “free-choice” trials. Inthe first “forced-choice” trial, the animal is confined 5 s in the startarm and then released while either the left or right goal arm is blockedby a horizontal door. After the mouse is released, it will negotiate themaze and eventually enter the open goal arm, and return to the startposition. Immediately after the return of the animal to the startposition, the closed goal door is opened and the animal is now free tochoose between the left and right goal arm (“free choice trials”). Theanimal is considered as entered when it places its four paws in the arm.A session is terminated and the animal is removed from the maze as soonas 14 free-choice trials have been performed or 10 min have elapsed,whatever event occurs first. The average duration of a trial is 6 min.

The apparatus is cleaned between each animal using alcohol (70°). Urineand feces are removed from the maze.

During the trials, animal handling and the visibility of the operatorare minimized as much as possible.

The percentage of alternation over the 14 free-choice trials wasdetermined for each mouse and was used as an index of working memoryperformance. This percentage was defined as entry in a different arm ofthe T-maze over successive trials (i.e., left-right-left-right, etc).

Statistical Analysis

Analysis of variance (ANOVA) was performed on the result data. Fisher'sProtected Least Significant Difference was used for pairwisecomparisons. p value≤0.05 were considered significant. The drug inducedreversion of scopolamine-induced memory deficit was calculated bysetting the respective response of the saline/vehicle group as 100% andthe scopolamine/vehicle group as 0% reversion.

In order to determine the ED₅₀ for each drug, the recovery performancewas plotted following a sigmoidal dose-response model (graphpadsoftware). ED50 was read from the curve fitting table and represents theeffective dose associated with 50% of response.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of any appended claims. All figures, tables, and appendices, aswell as publications, patents, and patent applications, cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method for the treatment of aneurodegenerative, psychiatric or neurological disease associated with acholinergic deficit comprising administering GLN-1062:

or a pharmaceutically acceptable salt thereof by nasal administration toa patient in need thereof.
 2. The method of claim 1, wherein the diseaseis selected from the group consisting of Alzheimer's disease,Parkinson's disease, other types of dementia, schizophrenia, epilepsy,stroke, poliomyelitis, neuritis, oxygen and nutrient deficiencies in thebrain after hypoxia, anoxia, asphyxia, cardiac arrest, chronic fatiguesyndrome, various types of poisoning, anesthesia, spinal cord disorders,central nervous system inflammation, postoperative delirium and/orsubsyndronal postoperative delirium, neuropathic pain, subsequences ofthe abuse of alcohol and drugs, addictive alcohol and nicotine craving,and subsequences of radiotherapy.
 3. The method of claim 1, wherein theneurodegenerative, psychiatric or neurological disease is selected fromthe group consisting of Alzheimer's disease, Parkinson's disease,dementia, schizophrenia, stroke, central nervous system inflammation andepilepsy.
 4. The method of claim 1, wherein GLN-1062 is administered ina pharmaceutical composition as a suspension.
 5. The method of claim 1,wherein GLN-1062 is administered in a pharmaceutical composition as asolution.
 6. The method of claim 4, wherein the suspension comprises 0.5to 30 wt % GLN-1062.
 7. The method of claim 5, wherein the solutioncomprises 0.5 to 30 wt % GLN-1062.
 8. The method of claim 1, comprisingmultiple doses to the patient, wherein a single dose comprises between0.1 and 20 mg of GLN-1062.